F Libert’s research while affiliated with Université Libre de Bruxelles and other places

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Publications (65)


Transcriptomic signature of human embryonic thyroid reveals transition from differentiation to functional maturation
  • Article

September 2022

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33 Reads

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3 Citations

Yearbook of Paediatric Endocrinology

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Dmitriev P

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Figure 2. Live imaging of transgenic Tg(tg:nlsEGFP) fish reveals diminished reporter expression and thyroid hypoplasia in thyroxine-treated fish. (A) Model for thyroid hormone negative feedback loop along pituitarythyroid axis with tissue-specific gene expression markers examined in this study. T4, thyroxine; TSH, thyroidstimulating hormone. (B,C) Treatment of zebrafish embryos with 5.0 µg/L T4 affects pituitary and thyroid gene expression as demonstrated by WISH. Pituitary expression of tshb mRNA (see ventral views in B) is reduced in T4-treated fish compared to untreated controls (Ct). At the thyroid level, expression of the TSH-dependent gene slc5a5 is strongly decreased following T4 treatment (ventral views in C, upper panels). T4-treated fish also showed decreased tg mRNA expression and a reduced number of tg mRNA expressing cells (ventral views in C, lower panels), effects which were more marked at 100 hpf. (D) Epifluorescence microscopy of live Tg(tg:nlsEGFP) fish revealed a strongly reduced intensity of the fluorescent reporter signal in T4-treated fish relative to untreated controls (Ct). For each embryo shown (ventral view), three-fold magnified views of the thyroid region are displayed (GFP channel). (E) Comparative analysis of thyroid morphology in control (Ct) and T4-treated larvae by confocal live imaging of g(tg:nlsEGFP) fish. 3D reconstructions of confocal images (ventral views) are shown. At 6 dpf, thyroids of T4-treated fish appeared atrophic (smaller follicles and reduced reporter expression). (F) Quantification of thyroid follicular cell (TFC) number in untreated controls (Ct) and T4-treated fish. Results are shown as means ± S.E.M (N = X − Y). Asterisks denote significant differences between treatment means at the indicated developmental stages (*P < 0.05, **P < 0.01, Student's t-test). Anterior is to the top in all images. Scale bars: 20 µm (B,E) and 100 µm (C,D). 
Figure 3. Recovery of thyroid dysgenesis phenotypes in zebrafish pax2a crispants. (A) Epifluorescence live imaging of Tg(tg:nlsEGFP) zebrafish. Ventral views of the head region (anterior to the right, scale bar: 100 µm) and magnified views of the thyroid region (GFP channel only, scale bar: 50 µm) are shown for non-injected controls and pax2a crispants at 55 hpf, 80 hpf, and 6 dpf. Phenotypic pax2a crispants displayed athyreosis or severe hypoplasia. (B) Whole-mount immunofluorescence staining of Tg(tg:nlsEGFP) zebrafish (6 dpf) for EGFP (thyroid cells) and thyroxine (colloidal T4). Epifluorescence imaging of the thyroid region in 6 dpf larvae (ventral views, anterior to the right, scale bar: 100 µm) revealed variable but reduced T4 content in pax2a crispants with thyroid hypoplasia and confirmed the absence of detectable T4 synthesis in pax2a crispants lacking EGFP-tagged thyroid cells (athyreosis group). (C) Distribution of allelic variants as determined by Illumina HiSeq analysis of individual pax2a crispants confirmed high mutagenesis efficiency in larvae affected by thyroid dysgenesis. The percentage of WT alleles (no variant call), in-frame indels, or frameshift indels is shown for N = 4 larvae per phenotypic category (median values with interquartile range). 
Figure 4. Recovery of dyshormonogenesis phenotypes in zebrafish duox crispants. (A) Epifluorescence live imaging of Tg(tg:nlsEGFP) zebrafish. Ventral views of the head region (anterior to the right, scale bar: 100 µm) and magnified views of the thyroid region (GFP channel only, scale bar: 50 µm) are shown for non-injected controls and duox crispants at 55 hpf, 80 hpf, and 6 dpf. No thyroid phenotypes were detectable at 55 hpf and 80 hpf but by 6 dpf, the majority of duox crispants displayed a goitrous thyroid enlargement with severe hyperplasia. (B) Whole-mount immunofluorescence staining of Tg(tg:nlsEGFP) zebrafish (6 dpf) for EGFP (thyroid cells) and thyroxine (colloidal T4). Epifluorescence imaging of the thyroid region in 6 dpf larvae (ventral views, anterior to the right, scale bar: 50 µm) revealed reduced T4 content in duox crispants as the most prevalent phenotypic alterations. Most of these hypothyroid larvae displayed concurrent thyroid hyperplasia. (C) Distribution of allelic variants as determined by Illumina HiSeq analysis of individual duox crispants confirmed high mutagenesis efficiency in larvae affected by thyroid dyshormonogenesis. The percentage of WT alleles (no variant call), in-frame indels, or frameshift indels is shown for N = 4 larvae per phenotypic category (median values with interquartile range). 
Figure 5. Recovery of hypoplastic/atrophic thyroid phenotypes in zebrafish tshr crispants. (A) Epifluorescence live imaging of Tg(tg:nlsEGFP) zebrafish. Ventral views of the head region (anterior to the right, scale bar: 100 µm) and magnified views of the thyroid region (GFP channel only, scale bar: 50 µm) are shown for noninjected controls and tshr crispants (target: exon 4) at 55 hpf, 80 hpf, and 6 dpf. No thyroid phenotypes were detectable at 55 hpf and 80 hpf but by 6 dpf, tshr crispants presented a hypoplastic/atrophic thyroid phenotype. (B) Whole-mount immunofluorescence staining of Tg(tg:nlsEGFP) zebrafish (6 dpf) for EGFP (thyroid cells) and thyroxine (colloidal T4). Epifluorescence imaging of the thyroid region in 6 dpf larvae (ventral views, anterior to the right, scale bar: 50 µm) revealed that thyroid hypoplasia in tshr crispants was accompanied by a reduction in thyroidal T4 content. (C) Distribution of allelic variants as determined by Illumina HiSeq analysis of individual tshr crispants revealed high mutagenesis efficiency in hypothyroid larvae presenting a hypoplastic thyroid. The percentage of WT alleles (no variant call), in-frame indels, or frameshift indels is shown for N = 4-6 larvae per phenotypic category (median values with interquartile range). 
Figure 6. tshr crispants are resistant to PTU-induced thyroid hyperplasia. (A) Confocal live imaging of Tg(tg:nlsEGFP) zebrafish at 6 dpf. Three-dimensional reconstructions of confocal images of the thyroid region (ventral views, anterior to the left, scale bars: 40 µm) are shown for tshr crispants and non-injected siblings raised in the presence or absence of 30 mg/L phenylthiourea (PTU). Non-injected control larvae developed severe thyroid hyperplasia in response to PTU treatment. In contrast, only about 50% of the PTU-treated tshr crispants displayed a similar thyroid enlargement (phenotype #2). The remaining tshr crispants showed an almost complete resistance to PTU-induced thyroid enlargement and instead presented with a hypoplastic/ atrophic thyroid phenotype (phenotype #1) similar to that seen in tshr crispants raised in the absence of PTU. (B) Quantification of thyroid follicular cell number in the different phenotypic groups at 6 dpf. Means ± S.E.M. are shown (N = 12-16). Different letters indicate significant differences between groups (P < 0.05, Tukey's multiple comparison test). (C) Confocal live imaging identified mosaicism in cellular responses to PTU treatment in several tshr crispants. Note the atrophic characteristics of the follicle in the middle (low reporter expression, flat epithelium) compared to the marked hypertrophy/hyperplasia of the two neighbouring follicles. 

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A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function
  • Article
  • Full-text available

April 2018

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564 Reads

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46 Citations

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

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Hyperosmotic stress induces cell cycle arrest in retinal pigmented epithelial cells

June 2013

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114 Reads

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34 Citations

Cell Death and Disease

Osmotic changes occur in many tissues and profoundly influence cell function. Herein, we investigated the effect of hyperosmotic stress on retinal pigmented epithelial (RPE) cells using a microarray approach. Upon 4-h exposure to 100 mM NaCl or 200 mM sucrose, 79 genes were downregulated and 72 upregulated. Three gene ontology categories were significantly modulated: cell proliferation, transcription from RNA polymerase II promoter and response to abiotic stimulus. Fluorescent-activated cell sorting analysis further demonstrated that owing to hyperosmotic stimulation for 24 h, cell count and cell proliferation, as well as the percentage of cells in G0/G1 and S phases were significantly decreased, whereas the percentage of cells in G2/M phases increased, and apoptosis and necrosis remained unaffected. Accordingly, hyperosmotic conditions induced a decrease of cyclin B1 and D1 expression, and an activation of the p38 mitogen-activated protein kinase. In conclusion, our results demonstrate that hypertonic conditions profoundly affect RPE cell gene transcription regulating cell proliferation by downregulation cyclin D1 and cyclin B1 protein expression.



La famille des récepteurs couplés aux protéines G et ses orphelins

February 2013

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14 Reads

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2 Citations

Medecine sciences: M/S

Les recepteurs couples aux proteines G partagent la meme organisation structurelle, repondent a une grande diversite de ligands et participent a la modulation des enzymes intracellulaires et des canaux ioniques par l'intermediaire des proteines G heterotrimeriques. De nombreuses strategies de criblage ont conduit au clonage de leurs genes qui forment, chez les mammiferes, une des plus grandes familles geniques: un grand nombre est caracterise par les proprietes pharmacologiques. Il reste cependant un certain nombre de recepteurs dits «orphelins» car ils n'ont pu etre attribues a des sous-familles connues, ni ne definissent de nouvelles sous-familles. C'est par l'etude de leur structure et de leur distribution tissulaire qu'on a pu en reconnaitre certains. Les recepteurs restes orphelins constituent potentiellement l'un des elements de nouveaux systemes de communication intercellulaire et permettent a l'industrie pharmaceutique d'aborder des voies de recherche originales


Génétique moléculaire des récepteurs olfactifs

February 2013

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14 Reads

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2 Citations

Medecine sciences: M/S

Les genes codant pour les recepteurs olfactifs ont ete isoles a partir d'une banque d'ADNc de la muqueuse olfactive. Ils constituent une famille tres etendue (plusieurs centaines de genes) codant pour des recepteurs a sept passages transmembranaires couples, par l'intermediaire de proteines G, a l'activation de l'adenylyl cyclase et de la phospholipase C. La grande diversite des recepteurs olfactifs reside surtout dans les domaines transmembranaires III, IV, V et VI, qui portent les sites de liaison des ligands, les molecules odorantes, et repond sans doute a la diversite des ligands




Citations (39)


... During embryo development, these cells come from different places of blastoderm and develop in different period [12]. As is known, thyroid gland development mainly contains three processes [13]. First, the thyroid anlage appears at a 3-week human embryo. ...

Reference:

Ectopic thyroid tissue in the breast: A case report
Transcriptomic signature of human embryonic thyroid reveals transition from differentiation to functional maturation
  • Citing Article
  • September 2022

Yearbook of Paediatric Endocrinology

... To identify potential conservation between the endostyle and vertebrate tissues, we reanalyzed the published single-cell dataset from zebrafish (Danio rerio) (30,(38)(39)(40). The results indicated that the embryonic lineages of zebrafish, including the pharyngeal endoderm, paraxial mesoderm, neural crest, and pharyngeal arch, exhibited high similarity in expression compared with the endostyle of S. clava (Fig. 1F), suggesting substantial similarity between the ectodermderived tissues from zebrafish and the endostyle. ...

Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland
  • Citing Article
  • September 2021

Yearbook of Paediatric Endocrinology

... This process typically takes over 4-6 months, enabling the study of gene function across generations and the longterm effects of gene disruption [35]. On the other hand, transient knockouts, or CRISPants, allow for rapid phenotype observation in injected F0 mosaic embryos, generating biallelic mutations in a high proportion of cells, enabling researchers to detect gene effects within hours or days [59][60][61][62][63][64][65]. ...

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function

... Chemokine receptors typically signal via the G αi signaling pathway (Patel et al., 2013). The atypical chemokine receptor 3 (ACKR3), initially described as an orphan receptor (Libert et al, 1990), was discovered to act as second high-affinity receptor for CXCL12, next to CXCR4, and was subsequently renamed to chemokine receptor CXCR7 (Balabanian et al, 2005). Extensive cell signaling and structural biology studies have now provided evidence that ACKR3 does not signal via the canonical G protein pathway of chemokine receptors, but only through -arrestin recruitment , Rajagopal et al., 2010, Kleist et al., 2022, Yen et al., 2022, and therefore belongs to the family of atypical chemokine receptors (Nibbs and Graham, 2013, Sarma et al., Due to the relevance of ACKR3 in cancer and multiple sclerosis (Scholten et al., 2015;Wang et al., 2018, Williams et al., 2014, Torphy et al., 2022, the search for different ACKR3 modulators has so far resulted in a variety of modalities that can target ACKR3, including e.g. ...

COMPLETE NUCLEOTIDE-SEQUENCE OF A PUTATIVE-G PROTEIN COUPLED RECEPTOR - RDC1
  • Citing Article
  • April 1990

Nucleic Acids Research

... Intriguingly, approximately 1% of the human Caucasian population is homozygous for a mutation in the CCR5 gene, consisting of a 32-base pair deletion (CCR5-∆32) that in turn leads to mis-folding of the protein and its subsequent absence from the cell surface. People carrying this mutation are highly resistant to HIV infection [17,18] and fall in the category of Exposed Seronegative Individuals (ESN). ...

Resistance to HIV1 infection in caucasian individuals bearing mutant alleles of the CCR5 chemokine
  • Citing Article
  • January 1996

Nature

... Наши результаты сопоставимы с результатами, полученными в Японии (Narumi S. и соавт. [ [28] путем гибридизации in situ локализовали его до 14q31. Ген содержит 10 экзонов, состоит из α-и β-субъединиц, соединенных дисульфидной связью. ...

Localization of human thyrotropin receptor gene to chromosome region 14q31 by in situ hybridization
  • Citing Article
  • January 1990

Cytogenetic and Genome Research

... Hyperosmolarity also leads to neoangiogenesis [6,7]. The retina cannot metabolize this excess lactate, resulting in acidic intracellular pH, synaptic dysfunction, extracellular deposition (drusen), and ultimately apoptosis [8]. Therefore, we suggest that AMD can be viewed as a mitochondrial failure secondary to inflammation and metabolic disease. ...

Hyperosmotic stress induces cell cycle arrest in retinal pigmented epithelial cells

Cell Death and Disease

... Während der GTP-Bindung bleibt die Adenylatzyklase aktiviert. Die Adenylatzyklase bildet mehrfach cAMP, das als Botenstoff (second messenger) im weiteren Verlauf eine Proteinkinase aktiviert, welche nun weitere Enzyme phosphoryliert [82]. ...

Molecular Genetics of the Thyrotropin Receptor
  • Citing Article
  • February 1992

Experimental and Clinical Endocrinology

... Among the four adenosine receptors, A 1 AR was the first adenosine receptor identified by pharmacological analysis in 1979 [13] and was cloned from dog in 1991 [14]. A 1 AR is widely expressed in the central nervous system and in peripheral tissue mediating protective effects against brain injury and ischemic kidney injury. ...

The orphan receptor cDNA RDC7 encodes an A1 adenosine receptor

The EMBO Journal

... Cloning has allowed the confirmation of the coexistence of both receptors in all the mammalian species studied so far. The first clone isolated was termed the dog RDC4 receptor (40), later identified as the 5-HT 1D receptor (41). Cloning and expression of the two receptors in the human (42-48) revealed a low amino acid similarity between them, with a very similar ligand-binding profile. ...

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor
  • Citing Article
  • December 1991

Biochemical and Biophysical Research Communications