Touma, A;Vang, D;Tang, W;Rasicci, D;Rai, A;Previs, S;Warshaw, D;Yengo, C;Sivaramakrishnan, S;
| DOI: 10.1016/j.bpj.2021.11.1468
Cardiac myosin-binding protein C (cMyBP-C) is an important regulator of cardiac muscle contraction and is commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanism of regulation by cMyBP-C remains unclear due to experimental challenges in dissecting the proposed weak, transient interactions with its binding partners. Here, we utilized a nanosurf assay, containing a synthetic b-cardiac myosin thick filament, to systematically probe cMyBP-C interactions with actin and myosin. Recombinant human b-cardiac myosin subfragments (HMM or S1) were attached to DNA nanotubes, with 14 or 28 nm spacing, similar to the myosin head spacing on native thick filaments. No significant difference in thin filament velocity was observed with 14 nm vs. 28 nm motor spacing. Various N-terminal fragments of cMyBP-C were interdigitated with b-cardiac myosin on DNA nanotubes via encoded SNAP tags labeled with sequence-specific oligo attachment strands. We recapitulated inhibition of thin filament motility on b-cardiac myosin HMM and S1 nanotubes by C0-C2 (4-6 fold) and C1-C2 (4-8 fold) N-terminal cMyBP-C fragments. Equivalent inhibition of b-cardiac myosin HMM and S1 subfragments suggests the actin-cMyBP-C interaction dominates this inhibitory mechanism. We found that a C0-C1f fragment lacking the majority of the M-domain did not inhibit b-cardiac myosin nanotube motility, confirming the importance of the M-domain in regulatory interactions. Diminished inhibition by C0-C2 and C1-C2 phosphomimetic fragments (2-3 fold higher velocity compared to their phosphonull counterparts) further highlights the importance of the phosphorylatable serines in the regulatory Mdomain. These results shed light on the mechanism of cMyBP-C and highlight the utility of the nanosurf as
Non-thermal plasma promotes hair growth by improving the inter-follicular macroenvironment
Kim, H;Choi, E;Choi, E;Kim, H;Kim, J;Cho, G;Kim, H;Na, S;Shin, J;Do, S;Park, B;
| DOI: 10.1039/d1ra04625j
Non-thermal plasma (NTP) is widely used in the disinfection and surface modification of biomaterials.
Mertz, E;Makareeva, E;Mirigian, L;Leikin, S;
| DOI: 10.1002/jbm4.10701
Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.
bioRxiv : the preprint server for biology
Martini, AG;Smith, JP;Medrano, S;Sheffield, NC;Sequeira-Lopez, MLS;Gomez, RA;
PMID: 36711565 | DOI: 10.1101/2023.01.18.524595
Renin cells are essential for survival. They control the morphogenesis of the kidney arterioles, and the composition and volume of our extracellular fluid, arterial blood pressure, tissue perfusion, and oxygen delivery. It is known that renin cells and associated arteriolar cells descend from FoxD1 + progenitor cells, yet renin cells remain challenging to study due in no small part to their rarity within the kidney. As such, the molecular mechanisms underlying the differentiation and maintenance of these cells remain insufficiently understood.We sought to comprehensively evaluate the chromatin states and transcription factors (TFs) that drive the differentiation of FoxD1 + progenitor cells into those that compose the kidney vasculature with a focus on renin cells.We isolated single nuclei of FoxD1 + progenitor cells and their descendants from FoxD1 cre/+ ; R26R-mTmG mice at embryonic day 12 (E12) (n cells =1234), embryonic day 18 (E18) (n cells =3696), postnatal day 5 (P5) (n cells =1986), and postnatal day 30 (P30) (n cells =1196). Using integrated scRNA-seq and scATAC-seq we established the developmental trajectory that leads to the mosaic of cells that compose the kidney arterioles, and specifically identified the factors that determine the elusive, myo-endocrine adult renin-secreting juxtaglomerular (JG) cell. We confirm the role of Nfix in JG cell development and renin expression, and identified the myocyte enhancer factor-2 (MEF2) family of TFs as putative drivers of JG cell differentiation.We provide the first developmental trajectory of renin cell differentiation as they become JG cells in a single-cell atlas of kidney vascular open chromatin and highlighted novel factors important for their stage-specific differentiation. This improved understanding of the regulatory landscape of renin expressing JG cells is necessary to better learn the control and function of this rare cell population as overactivation or aberrant activity of the RAS is a key factor in cardiovascular and kidney pathologies.
American journal of human genetics
Khalaf-Nazzal, R;Fasham, J;Inskeep, KA;Blizzard, LE;Leslie, JS;Wakeling, MN;Ubeyratna, N;Mitani, T;Griffith, JL;Baker, W;Al-Hijawi, F;Keough, KC;Gezdirici, A;Pena, L;Spaeth, CG;Turnpenny, PD;Walsh, JR;Ray, R;Neilson, A;Kouranova, E;Cui, X;Curiel, DT;Pehlivan, D;Akdemir, ZC;Posey, JE;Lupski, JR;Dobyns, WB;Stottmann, RW;Crosby, AH;Baple, EL;
PMID: 36283405 | DOI: 10.1016/j.ajhg.2022.09.012
Non-centrosomal microtubules are essential cytoskeletal filaments that are important for neurite formation, axonal transport, and neuronal migration. They require stabilization by microtubule minus-end-targeting proteins including the CAMSAP family of molecules. Using exome sequencing on samples from five unrelated families, we show that bi-allelic CAMSAP1 loss-of-function variants cause a clinically recognizable, syndromic neuronal migration disorder. The cardinal clinical features of the syndrome include a characteristic craniofacial appearance, primary microcephaly, severe neurodevelopmental delay, cortical visual impairment, and seizures. The neuroradiological phenotype comprises a highly recognizable combination of classic lissencephaly with a posterior more severe than anterior gradient similar to PAFAH1B1(LIS1)-related lissencephaly and severe hypoplasia or absence of the corpus callosum; dysplasia of the basal ganglia, hippocampus, and midbrain; and cerebellar hypodysplasia, similar to the tubulinopathies, a group of monogenic tubulin-associated disorders of cortical dysgenesis. Neural cell rosette lineages derived from affected individuals displayed findings consistent with these phenotypes, including abnormal morphology, decreased cell proliferation, and neuronal differentiation. Camsap1-null mice displayed increased perinatal mortality, and RNAScope studies identified high expression levels in the brain throughout neurogenesis and in facial structures, consistent with the mouse and human neurodevelopmental and craniofacial phenotypes. Together our findings confirm a fundamental role of CAMSAP1 in neuronal migration and brain development and define bi-allelic variants as a cause of a clinically distinct neurodevelopmental disorder in humans and mice.
Contemporary Clinical Neuroscience
Rahimi-Balaei, M;Ramirez, M;Gupta, I;Goldowitz, D;
| DOI: 10.1007/978-3-031-23104-9_6
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International journal of molecular sciences
Abou Nader, N;Zamberlam, G;Boyer, A;
PMID: 36430866 | DOI: 10.3390/ijms232214388
The cortex of the adrenal gland is organized into concentric zones that produce distinct steroid hormones essential for body homeostasis in mammals. Mechanisms leading to the development, zonation and maintenance of the adrenal cortex are complex and have been studied since the 1800s. However, the advent of genetic manipulation and transgenic mouse models over the past 30 years has revolutionized our understanding of these mechanisms. This review lists and details the distinct Cre recombinase mouse strains available to study the adrenal cortex, and the remarkable progress total and conditional knockout mouse models have enabled us to make in our understanding of the molecular mechanisms regulating the development and maintenance of the adrenal cortex.
Andersen RE, Hong SJ, Lim JJ, Cui M, Harpur BA, Hwang E, Delgado RN, Ramos AD, Liu SJ, Blencowe BJ, Lim DA.
PMID: 31112699 | DOI: 10.1016/j.devcel.2019.04.032
While it is now appreciated that certain long noncoding RNAs (lncRNAs) have important functions in cell biology, relatively few have been shown to regulate development in vivo, particularly with genetic strategies that establish cis versus trans mechanisms. Pnky is a nuclear-enriched lncRNA that is transcribed divergently from the neighboring proneural transcription factor Pou3f2. Here, we show that conditional deletion of Pnky from the developing cortex regulates the production of projection neurons from neural stem cells (NSCs) in a cell-autonomous manner, altering postnatal cortical lamination. Surprisingly, Pou3f2 expression is not disrupted by deletion of the entire Pnky gene. Moreover, expression of Pnky from a BAC transgene rescues the differential gene expression and increased neurogenesis of Pnky-knockout NSCs, as well as the developmental phenotypes of Pnky-deletion in vivo. Thus, despite being transcribed divergently from a key developmental transcription factor, the lncRNA Pnky regulates development in trans
Marcy, G;Foucault, L;Babina, E;Capeliez, T;Texeraud, E;Zweifel, S;Heinrich, C;Hernandez-Vargas, H;Parras, C;Jabaudon, D;Raineteau, O;
PMID: 37146152 | DOI: 10.1126/sciadv.abq7553
The ventricular-subventricular zone (V-SVZ) is the largest neurogenic region of the postnatal forebrain, containing neural stem cells (NSCs) that emerge from both the embryonic pallium and subpallium. Despite of this dual origin, glutamatergic neurogenesis declines rapidly after birth, while GABAergic neurogenesis persists throughout life. We performed single-cell RNA sequencing of the postnatal dorsal V-SVZ for unraveling the mechanisms leading to pallial lineage germinal activity silencing. We show that pallial NSCs enter a state of deep quiescence, characterized by high bone morphogenetic protein (BMP) signaling, reduced transcriptional activity and Hopx expression, while in contrast, subpallial NSCs remain primed for activation. Induction of deep quiescence is paralleled by a rapid blockade of glutamatergic neuron production and differentiation. Last, manipulation of Bmpr1a demonstrates its key role in mediating these effects. Together, our results highlight a central role of BMP signaling in synchronizing quiescence induction and blockade of neuronal differentiation to rapidly silence pallial germinal activity after birth.
Goodwin, K;Lemma, B;Zhang, P;Boukind, A;Nelson, CM;
PMID: 36868232 | DOI: 10.1016/j.devcel.2023.02.002
It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.
Labbaf, Z;Petratou, K;Ermlich, L;Backer, W;Tarbashevich, K;Reichman-Fried, M;Luschnig, S;Schulte-Merker, S;Raz, E;
PMID: 35914525 | DOI: 10.1016/j.devcel.2022.07.008
Cell ablation is a key method in the research fields of developmental biology, tissue regeneration, and tissue homeostasis. Eliminating specific cell populations allows for characterizing interactions that control cell differentiation, death, behavior, and spatial organization of cells. Current methodologies for inducing cell death suffer from relatively slow kinetics, making them unsuitable for analyzing rapid events and following primary and immediate consequences of the ablation. To address this, we developed a cell-ablation system that is based on bacterial toxin/anti-toxin proteins and enables rapid and cell-autonomous elimination of specific cell types and organs in zebrafish embryos. A unique feature of this system is that it uses an anti-toxin, which allows for controlling the degree and timing of ablation and the resulting phenotypes. The transgenic zebrafish generated in this work represent a highly efficient tool for cell ablation, and this approach is applicable to other model organisms as demonstrated here for Drosophila.
Development (Cambridge, England)
Price, JD;Lindtner, S;Ypsilanti, A;Binyameen, F;Johnson, JR;Newton, BW;Krogan, NJ;Rubenstein, JLR;
PMID: 35695185 | DOI: 10.1242/dev.199508
In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.
