Publications by authors named "Alessia Gambadoro"

8 Publications

  • Page 1 of 1

Functional loss of leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.

Dis Model Mech 2019 08 2;12(8). Epub 2019 Aug 2.

European Mouse Mutant Archive (EMMA), INFRAFRONTIER, Monterotondo Mouse Clinic, Department of Biomedical Sciences (DSB), Italian National Research Council (CNR), Adriano Buzzati-Traverso Campus, via Ramarini, 32, 00015, Monterotondo, Rome, Italy.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the loss-of-function mutation ( mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151-β-galactosidase reporter expression in whole-mount brain and histological analysis show that is expressed in ependymal cells lining the ventricular brain system, further confirming the role of dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the -knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the -knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in null animals. gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper.
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http://dx.doi.org/10.1242/dmm.038489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737950PMC
August 2019

Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals.

Mamm Genome 2018 04 23;29(3-4):245-259. Epub 2017 Nov 23.

Institute of Cell Biology and Neurobiology (IBCN), via Ramarini, 32, 00015, Monterotondo, Rome, Italy.

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.
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http://dx.doi.org/10.1007/s00335-017-9723-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887010PMC
April 2018

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Nat Genet 2015 Sep 27;47(9):969-978. Epub 2015 Jul 27.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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http://dx.doi.org/10.1038/ng.3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564951PMC
September 2015

Dissection of the carboxyl-terminal domain of the proteasomal subunit Rpn11 in maintenance of mitochondrial structure and function.

Mol Biol Cell 2008 Mar 2;19(3):1022-31. Epub 2008 Jan 2.

Pasteur Institute-Cenci Bolognetti Foundation, Department of Cell and Developmental Biology, University of Rome La Sapienza, 00185 Rome, Italy.

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN+/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative alpha-helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes.
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http://dx.doi.org/10.1091/mbc.e07-07-0717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2262987PMC
March 2008

Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

Biochem J 2004 Jul;381(Pt 1):275-85

Pasteur Institute Cenci Bolognetti Foundation and the Department of Cell and Developmental Biology, University of Rome I, 00185 Rome, Italy.

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.
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http://dx.doi.org/10.1042/BJ20040008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133786PMC
July 2004