Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Ectopic expression of CITED2 prior to reprogramming, promotes and homogenises the conversion of somatic cells into induced pluripotent stem cells.

Cited2 plays crucial roles in mouse embryonic stem cells self-renewal, the initiation of the somatic reprogramming process into induced pluripotent stem cells (iPSC) and the suppression of cell senescence. Here, we investigated the potential of CITED2 expression in combination with the Oct4, Sox2, Klf4 and c-Myc factors for reprogramming of primary mouse embryonic fibroblasts (MEF) at passage 2 and 4. The ectopic CITED2 expression in primary MEF prior to the onset of the reprogramming process, generated iPSC with less variability in the expression of endogenous pluripotency-related genes. In contrast, part of the MEF reprogrammed without ectopic expression of CITED2 at passage 4 originated partially reprogrammed iPSC or pre-iPSC. However, the overexpression of CITED2 in the pre-iPSC was insufficient to complete the reprogramming process into iPSC. These results indicated that ectopic CITED2 expression at the onset of the reprogramming process in combination with the reprogramming factors promotes a complete and homogeneous conversion of somatic cells into iPSC.

Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants.

Mutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD.

CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells.

The transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.