Biology subjects

Astrof, S.

Publications and source records attributed to Astrof, S..

9 recordsLinked to original sources

bioRxiv2024

A screen of mutants generated and imaged by the International Mouse Phenotyping Consortium identifies 39 novel genes regulating the development of the secondary palate.

The International Mouse Phenotyping Consortium (IMPC) has generated thousands of knockout mouse lines, many of which exhibit embryonic or perinatal lethality. Using micro-computed tomography (micro-CT), the IMPC has created and publicly released 3D image datasets of embryos from these lethal and subviable lines. In this study, we leveraged this dataset to screen homozygous null mutants for anomalies in secondary palate development. We analyzed optical sections from 2,987 embryos at embryonic days E15.5 and E18.5, representing 484 homozygous mutant lines. Our analysis identified 45 novel genes implicated in palatogenesis. Gene set enrichment analysis highlighted biological processes and pathways relevant to palate development and uncovered 18 genes jointly regulating the development of the eye and the palate. These findings present a valuable resource for further research, offer novel insights into the molecular mechanisms underlying palatogenesis, and provide important context for understanding the etiology of rare human congenital disorders involving simultaneous malformations of the palate and other organs, including the eyes, ears, kidneys, and lungs.

developmental biology↗

bioRxiv2023

SMAD4: A Critical Regulator of Cardiac Neural Crest Cell Fate and Vascular Smooth Muscle Differentiation

BackgroundThe pharyngeal arch arteries (PAAs) are precursor vessels which remodel into the aortic arch arteries (AAAs) during embryonic cardiovascular development. Cardiac neural crest cells (NCs) populate the PAAs and differentiate into vascular smooth muscle cells (vSMCs), which is critical for successful PAA-to-AAA remodeling. SMAD4, the central mediator of canonical TGF{beta} signaling, has been implicated in NC-to-vSMC differentiation; however, its distinct roles in vSMC differentiation and NC survival are unclear. ResultsHere, we investigated the role of SMAD4 in cardiac NC differentiation to vSMCs using lineage-specific inducible mouse strains in an attempt to avoid early embryonic lethality and NC cell death. We found that with global SMAD4 loss, its role in smooth muscle differentiation could be uncoupled from its role in the survival of the cardiac NC in vivo. Moreover, we found that SMAD4 may regulate the induction of fibronectin, a known mediator of NC-to-vSMC differentiation. Finally, we found that SMAD4 is required in NCs cell-autonomously for NC-to-vSMC differentiation and for NC contribution to and persistence in the pharyngeal arch mesenchyme. ConclusionsOverall, this study demonstrates the critical role of SMAD4 in the survival of cardiac NCs, their differentiation to vSMCs, and their contribution to the developing pharyngeal arches.

developmental biology↗

bioRxiv2023

Identification of novel buffering mechanisms in aortic arch artery development and congenital heart disease.

RationaleThe resiliency of embryonic development to genetic and environmental perturbations has been long appreciated; however, little is known about the mechanisms underlying the robustness of developmental processes. Aberrations resulting in neonatal lethality are exemplified by congenital heart disease (CHD) arising from defective morphogenesis of pharyngeal arch arteries (PAA) and their derivatives. ObjectiveTo uncover mechanisms underlying the robustness of PAA morphogenesis. Methods and ResultsThe second heart field (SHF) gives rise to the PAA endothelium. Here, we show that the number of SHF-derived ECs is regulated by VEGFR2 and Tbx1. Remarkably, when SHF-derived EC number is decreased, PAA development can be rescued by the compensatory endothelium. Blocking such compensatory response leads to embryonic demise. To determine the source of compensating ECs and mechanisms regulating their recruitment, we investigated three-dimensional EC connectivity, EC fate, and gene expression. Our studies demonstrate that the expression of VEGFR2 by the SHF is required for the differentiation of SHF-derived cells into PAA ECs. The deletion of one VEGFR2 allele (VEGFR2SHF-HET) reduces SHF contribution to the PAA endothelium, while the deletion of both alleles (VEGFR2SHF-KO) abolishes it. The decrease in SHF-derived ECs in VEGFR2SHF-HET and VEGFR2SHF-KO embryos is complemented by the recruitment of ECs from the nearby veins. Compensatory ECs contribute to PAA derivatives, giving rise to the endothelium of the aortic arch and the ductus in VEGFR2SHF-KO mutants. Blocking the compensatory response in VEGFR2SHF-KO mutants results in embryonic lethality shortly after mid-gestation. The compensatory ECs are absent in Tbx1+/- embryos, a model for 22q11 deletion syndrome, leading to unpredictable arch artery morphogenesis and CHD. Tbx1 regulates the recruitment of the compensatory endothelium in an SHF-non-cell-autonomous manner. ConclusionsOur studies uncover a novel buffering mechanism underlying the resiliency of PAA development and remodeling. Nonstandard Abbreviations and Acronyms in Alphabetical OrderCHD - congenital heart disease; ECs - endothelial cells; IAA-B - interrupted aortic arch type B; PAA - pharyngeal arch arteries; RERSA - retro-esophageal right subclavian artery; SHF - second heart field; VEGFR2 - Vascular endothelial growth factor receptor 2.

developmental biology↗

bioRxiv2022

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process

Fibronectin fibrillogenesis and mechanosensing both depend on integrin-mediated force transmission to the extracellular-matrix. However, force transmission is in itself dependent on fibrillogenesis, and fibronectin fibrils are found in soft embryos where high forces cannot be applied, suggesting that force cannot be the sole initiator of fibrillogenesis. Here we identify a nucleation step prior to force transmission, driven by fibronectin oxidation mediated by lysyl-oxidase enzyme family members. This oxidation induces fibronectin clustering that promotes early adhesion, alters cellular response to soft matrices, and enhances force transmission to the matrix. In contrast, absence of fibronectin oxidation abrogates fibrillogenesis, perturbs cell-matrix adhesion, and compromises mechanosensation. Moreover, fibronectin oxidation promotes cancer cells colony formation in soft agar as well as collective and single-cell migration. These results reveal a force-independent enzyme-dependent mechanism that initiates fibronectin fibrillogenesis, establishing a critical step in cell adhesion and mechanosensing.

cell biology↗

bioRxiv2022

The mesodermal source of fibronectin is required for heart morphogenesis and cardiac outflow tract elongation by regulating cell shape, polarity, and mechanotransduction in the second heart field.

Failure in the elongation of the cardiac outflow tract results in congenital heart disease due to ventricular septum defects and misalignment of the great vessels. The cardiac outflow tract lengthens via accretion of progenitors derived from the second heart field (SHF). SHF cells in the splanchnic mesoderm are exquisitely regionalized and organized into an epithelial-like layer forming the dorsal pericardial wall (DPW). Tissue tension, cell polarity, and proliferation within the DPW are important for the addition of SHF-derived cells to the heart and elongation of the cardiac outflow tract. However, the genes regulating these processes are not completely characterized. Using conditional mutagenesis in the mouse, we show that fibronectin (Fn1) synthesized by the SHF is a central regulator of epithelial architecture in the DPW. Fn1 is enriched in the anterior DPW and mediates outflow tract elongation by balancing pro- and anti-adhesive cell-ECM interactions and regulating DPW cell shape, polarity, cohesion, proliferation, and mechanoresponsiveness. Our studies establish that Fn1 synthesized specifically by the mesoderm coordinates multiple cellular behaviors in the anterior DPW necessary for elongation of the cardiac outflow tract.

developmental biology↗

bioRxiv2021

Integration of vascular progenitors into functional blood vessels represents a novel mechanism of vascular growth

During embryogenesis, the initial vascular network is thought to form by the process of vasculogenesis, or the specification of vascular progenitors de novo. After the initial blood circulation has been established, the majority of later-forming vessels are thought to arise by angiogenesis from the already established vasculature. Here we show that new vascular progenitors in zebrafish embryos contribute to functional vasculature even after blood circulation has been established. Based on the expression analysis of early vascular progenitor markers etv2 and tal1, we characterized a novel site of late vasculogenesis (termed secondary vascular field, SVF), located bilaterally along the yolk extension. Using time-lapse imaging of etv2 reporter lines, we show that SVF cells migrate and incorporate into functional blood vessels and contribute to the formation of the posterior cardinal vein and subintestinal vasculature, suggesting a novel mode of vascular growth. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of etv2 function prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. In addition, we performed single-cell RNA-seq analysis to identify the transcriptional profile of SVF cells, which demonstrated similarities and differences between the transcriptomes of SVF cells and early vascular progenitors. Our results characterize a novel mechanism of how new vascular progenitors incorporate into established vasculature and revise our understanding of basic mechanisms that regulate vascular development.

developmental biology↗

bioRxiv2020

New mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fn1 fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live imaging and single-molecule localization microscopy (SMLM) are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing 6-11 Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, wherein nanodomains are spaced at the average periodicity of 105{+/-}17 nm. Periodical Fn1 nanodomain arrays are bona fide fibrils: they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. FUD, a bacterial peptide that binds Fn1 N-terminus and disrupts Fn1 fibrillogenesis does not disrupt the formation of Fn1 nanodomains, instead, it blocks the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis.

cell biology↗

bioRxiv2020

Cell-ECM interactions play distinct and essential roles at multiple stages during the development of the aortic arch arteries

RationaleDefects in the morphogenesis of the 4th pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. ObjectiveCell-ECM interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery (AAA) and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin 5{beta}1 and fibronectin (Fn1) expressed in the Isl1 lineages regulate PAA formation. The objective of these studies was to investigate cellular mechanisms by which integrin 5{beta}1 and Fn1 regulate AAA morphogenesis. Methods and ResultsUsing temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and populate pharyngeal arch mesenchyme between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches become organized into a uniform plexus of small blood vessels, which becomes remodeled into the PAAs between 31 - 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on pharyngeal ECM microenvironment extrinsic to the endothelium. Conditional ablation of integrin 5{beta}1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates AAA morphogenesis at multiple steps: 1) the recruitment of the SHF-derived ECs into the pharyngeal arches, 2) the remodeling of the uniform EC plexus in the 4th arches into the PAAs; and 3) differentiation of neural crest-derived cells abutting the PAA endothelium into vascular smooth muscle cells. ConclusionsPAA formation is a multi-step process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the AAA and its major branches. Cell-ECM interactions regulated by integrin 5{beta}1 and Fn1 play essential roles at each of these developmental stages.

developmental biology↗

bioRxiv2019

H2A.Z-dependent and -independent recruitment of metabolic enzymes to chromatin required for histone modifications

H2A.Z plays a fundamental role in the regulation of transcription and epigenetics, however, the mechanisms that underlie its functions are not fully understood. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with an array of tricarboxylic acid cycle and beta-oxidation enzymes in the mouse heart. Recombinant green florescence fusion proteins combined with mutations of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and chromatin binding in both rodent and human cells. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start sites. Finally, human H2A.Z-deficient HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, with the exception of pyruvate dehydrogenase, accompanied with reduced posttranslational histone modifications. Thus, the data show that metabolic enzymes are recruited to active promoters for potential site-directed epigenetic modifications.

molecular biology↗