The fate of early perichondrial cells in developing bones

Nature communications

2022 Nov 28

Matsushita, Y;Chu, AKY;Tsutsumi-Arai, C;Orikasa, S;Nagata, M;Wong, SY;Welch, JD;Ono, W;Ono, N;
PMID: 36443296 | DOI: 10.1038/s41467-022-34804-6

In endochondral bone development, bone-forming osteoblasts and bone marrow stromal cells have dual origins in the fetal cartilage and its surrounding perichondrium. However, how early perichondrial cells distinctively contribute to developing bones remain unidentified. Here we show using in vivo cell-lineage analyses that Dlx5+ fetal perichondrial cells marked by Dlx5-creER do not generate cartilage but sustainably contribute to cortical bone and marrow stromal compartments in a manner complementary to fetal chondrocyte derivatives under the regulation of Hedgehog signaling. Postnatally, Dlx5+ fetal perichondrial cell derivatives preferentially populate the diaphyseal marrow stroma with a dormant adipocyte-biased state and are refractory to parathyroid hormone-induced bone anabolism. Therefore, early perichondrial cells of the fetal cartilage are destined to become an adipogenic subset of stromal cells in postnatal diaphyseal bone marrow, supporting the theory that the adult bone marrow stromal compartments are developmentally prescribed within the two distinct cells-of-origins of the fetal bone anlage.

GPC3-Unc5 receptor complex structure and role in cell migration

Cell

2022 Oct 13

Akkermans, O;Delloye-Bourgeois, C;Peregrina, C;Carrasquero-Ordaz, M;Kokolaki, M;Berbeira-Santana, M;Chavent, M;Reynaud, F;Raj, R;Agirre, J;Aksu, M;White, ES;Lowe, E;Ben Amar, D;Zaballa, S;Huo, J;Pakos, I;McCubbin, PTN;Comoletti, D;Owens, RJ;Robinson, CV;Castellani, V;Del Toro, D;Seiradake, E;
PMID: 36240740 | DOI: 10.1016/j.cell.2022.09.025

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.

Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain

eLife

2022 Feb 17

Lee, DR;Rhodes, C;Mitra, A;Zhang, Y;Maric, D;Dale, RK;Petros, TJ;
PMID: 35175194 | DOI: 10.7554/eLife.71864

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain.

Molecular analysis of AAV5-hFVIII-SQ vector genome-processing kinetics in transduced mouse and nonhuman primate livers

Molecular Therapy - Methods & Clinical Development

2021 Dec 01

Sihn, C;Handyside, B;Liu, S;Zhang, L;Murphy, R;Yates, B;Xie, L;Torres, R;Russell, C;O’Neill, C;Pungor, E;Bunting, S;Fong, S;
| DOI: 10.1016/j.omtm.2021.12.004

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation Factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissue. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and nonhuman primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and nonhuman primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.

Loss of renal olfactory receptor 1393 leads to improved glucose homeostasis in a type 1 diabetic mouse model

Physiological reports

2021 Dec 01

Schiazza, AR;Considine, EG;Betcher, M;Shepard, BD;
PMID: 34877823 | DOI: 10.14814/phy2.15007

Renal olfactory receptor 1393 (Olfr1393) is an understudied sensory receptor that contributes to glucose handling in the proximal tubule. Our previous studies have indicated that this receptor may serve as a regulator of the sodium glucose co-transporters (SGLTs) and contributes to the development of glucose intolerance and hyperfiltration in the setting of diet-induced obesity. We hypothesized that Olfr1393 may have a similar function in Type 1 Diabetes. Using Olfr1393 wildtype (WT) and knockout (KO) mice along with streptozotocin (STZ) to induce pancreatic β-cell depletion, we tracked the development and progression of diabetes over 12 weeks. Here we report that diabetic male Olfr1393 KO mice have a significant improvement in hyperglycemia and glucose tolerance, despite remaining susceptible to STZ. We also confirm that Olfr1393 localizes to the renal proximal tubule, and have uncovered additional expression within the glomerulus. Collectively, these data indicate that loss of renal Olfr1393 affords protection from STZ-induced type 1 diabetes and may be a general regulator of glucose handling in both health and disease.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications

Molecular and Cellular Endocrinology

2022 Jan 01

Pulawska, K;Ponikwicka-Tyszko, D;Lebiedzinska, W;Guo, P;Bernaczyk, P;Pilaszewicz-Puza, A;Li, X;Chrusciel, M;Lupu, O;Leskinen, S;Makela, J;Toppari, J;Wolczynski, S;Coelingh Bennink, H;Huhtaniemi, I;Rahman, N;
| DOI: 10.1016/j.mce.2021.111502

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a healthy pregnancy

Cellular and molecular gastroenterology and hepatology

2021 Aug 23

Lee, J;Garcia, V;Nambiar, SM;Jiang, H;Dai, G;
PMID: 34438112 | DOI: 10.1016/j.jcmgh.2021.08.009

Maternal liver exhibits robust adaptations to pregnancy to accommodate the metabolic needs of developing and growing placenta and fetus by largely unknown mechanisms. We found that achaete-scute homolog-like 1 (Ascl1), a gene encoding a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice.To investigate whether and how Ascl1 plays a pregnancy-dependent role, we deleted the Ascl1 gene specifically in maternal hepatocytes from mid-gestation until term.As a result, we identified multiple Ascl1-dependent phenotypes. Maternal livers lacking Ascl1 exhibited aberrant hepatocyte structure, increased hepatocyte proliferation, enlarged hepatocyte size, reduced albumin production, and elevated release of liver enzymes, indicating maternal liver dysfunction. Simultaneously, maternal pancreas and spleen and the placenta displayed marked overgrowth; and the maternal ceca microbiome showed alterations in relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1-deficient dams experienced abnormal postnatal growth after weaning, implying an adverse pregnancy outcome. Mechanistically, we found that maternal hepatocytes deficient for Ascl1 exhibited robust activation of insulin-like growth factor 2 expression, which may contribute to the Ascl1-dependent phenotypes widespread in maternal and uteroplacental compartments.In summary, we demonstrate that maternal liver, via activating Ascl1 expression, modulates the adaptations of maternal organs and the growth of the placenta to maintain a healthy pregnancy. Our studies reveal Ascl1 as a novel and critical regulator of the physiology of pregnancy.

New insights into molecular changes in skeletal muscle aging and disease: Differential alternative splicing and senescence

Mechanisms of ageing and development

2021 May 18

Solovyeva, E;Ibebunjo, C;Utzinger, S;Eash, JK;Dunbar, A;Naumann, U;Zhang, Y;Serluca, FC;Demirci, S;Oberhauser, B;Black, F;Rausch, M;Hoersch, S;Meyer, A;
PMID: 34019916 | DOI: 10.1016/j.mad.2021.111510

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice

Nature communications

2021 May 13

Hunt, C;Hartford, SA;White, D;Pefanis, E;Hanna, T;Herman, C;Wiley, J;Brown, H;Su, Q;Xin, Y;Voronin, D;Nguyen, H;Altarejos, J;Crosby, K;Haines, J;Cancelarich, S;Drummond, M;Moller-Tank, S;Malpass, R;Buckley, J;Del Pilar Molina-Portela, M;Droguett, G;Frendewey, D;Chiao, E;Zambrowicz, B;Gong, G;
PMID: 33986266 | DOI: 10.1038/s41467-021-22932-4

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development

Nat Commun

2020 Apr 29

Vinckier NK, Patel NA, Geusz RJ, Wang A, Wang J, Matta I, Harrington AR, Wortham M, Wetton N, Wang J, Jhala US, Rosenfeld MG, Benner CW4, Shih HP, Sander M
PMID: 32350257 | DOI: 10.1038/s41467-020-16017-x

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited

Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation

Developmental cell

2023 Mar 28

Wang, H;Wang, Z;Zhou, T;Morris, D;Chen, S;Li, M;Wang, Y;Zheng, H;Fu, W;Yan, W;
PMID: 37023748 | DOI: 10.1016/j.devcel.2023.03.010

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

Liver development during Xenopus tropicalis metamorphosis is controlled by T3-activation of WNT signaling.

iScience

2023 Feb 01

Tanizaki, Y;Wang, S;Zhang, H;Shibata, Y;Shi, Y;
| DOI: 10.1016/j.isci.2023.106301

Thyroid hormone (T3) regulates vertebrate organ development, growth, and metabolism through T3 receptor (TR). Due to maternal influence in mammals, it has been difficult to study if and how T3 regulates liver development. Liver remodeling during anuran metamorphosis resembles liver maturation in mammals and is controlled by T3. We generated Xenopus tropicalis animals with both TRα and TRβ genes knocked out and found that TR double knockout liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate urea cycle gene expression. RNA-seq analysis showed that T3 activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely promoted proliferation and maturation of hepatocytes. Our study offers new insights on not only how T3 regulates liver development but also potential means to improve liver regeneration.

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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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