A Zebrafish Model of Parn-Deficiency Reveals Conserved Mechanisms Underlying Marrow Failure and Opportunities for Therapeutic Amelioration of Hematopoiesis
Notice bibliographique
Résumé
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome (IBMFS) and a telomere biology disorder that is characterized by physical malformations such as nail dystrophy, oral leukoplakia, reticular hyperpigmentation, multilineage cytopenia and immunodeficiency. Definitive treatment for the hematological complications is hematopoietic stem cell transplantation (HSCT), which is associated with high risk of organ toxicity and engraftment failure in this population. Further, DC patients are prone to developing myelodysplastic syndrome (MDS) and solid tumors. Germline mutations in the poly(A)-specific ribonuclease (PARN) gene are associated with Hoyeraal-Hreidarsson syndrome, the most severe form of DC. PARN encodes a deadenylase enzyme involved in the 3'-end processing of various RNAs including the telomerase RNA component (TERC). The clinical heterogeneity of phenotypes and the paucity of primary human samples necessitate the use of animal models such as zebrafish ( Danio rerio), given their highly conserved genetic program, to elucidate underlying biology. Zebrafish have a single PARN ortholog with 64% sequence identity at the amino acid level. Using CRISPR-Cas9 mutagenesis, we generated a 1.3 kb deletion in the zebrafish parn gene, predicted to result in premature protein truncation. To measure the hematopoietic output of the parn mutants, we performed whole-mount in situ hybridization (WISH) for key hematopoietic lineage markers. At 48 hours post-fertilization (hpf), we found a reduction in lcp1 + total leukocytes, mpx + neutrophils and hbbe3 + mature erythrocytes. Impaired erythroid differentiation was accompanied by an accumulation of gata1 + erythroid progenitors. This finding was further corroborated by o-dianisidine staining, showing reduced hemoglobinized erythrocytes. Thus, erythrocyte and leukocyte differentiation are compromised in our zebrafish parn mutant, phenocopying the multilineage cytopenia observed in DC patients. Flow cytometry of the whole kidney marrow (WKM, human bone marrow equivalent) at 12 months of age revealed an increase in hematopoietic stem and progenitor cells in the parn mutants. Giemsa staining of WKM touchpreps further identified an increase in myeloid-biased progenitors. Next, we investigated the consequences of Parn loss on telomerase activity. Using the telomere repeat amplification protocol, we found reduced telomerase activity in the WKMs of parn mutant zebrafish. Using 3' rapid amplification of cDNA ends, we observed longer ends in the terc amplicons of the parn mutants, reminiscent of defective deadenylation of human TERC RNA in the setting of PARN mutations. Exogenous thymidine supplementation has been shown to support telomere elongation in human cells. Following treatment of zebrafish embryos with thymidine nucleoside, we observed an improvement in erythrocyte numbers in the parn mutants by o-dianisidine staining at 48 hpf. Collectively, our preliminary findings suggest that telomerase-dependent mechanisms may contribute to the erythropenia seen in parn-mutant zebrafish. In summary, we generated a robust zebrafish model carrying a germline parn mutation that phenocopies the molecular and hematologic features seen in DC patients. Our model holds promise as a new in vivo tool for investigating DC-related disease mechanisms and as a platform for preclinical therapeutic screening.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,001 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,005 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».