Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreERT2 transgene at the otx2 gene locus to generate a conditional Cre-driver line.We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreERT2 upstream of the endogenous ATG of otx2, we utilized this gene’s native promoter and enhancer elements to perfectly match CreERT2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreERT2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

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No description available.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.