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Background: Dand5 encodes a protein that acts as an antagonist to Nodal/TGF-β and Wnt pathways. A mouse knockout (KO) model has shown that this molecule is associated with left-right asymmetry and cardiac development, with its depletion causing heterotaxia and cardiac hyperplasia. Objective: This study aimed to investigate the molecular mechanisms affected by the depletion of Dand5. Methods: DAND5-KO and wild-type embryoid bodies (EBs) were used to assess genetic expression with RNA sequencing. To complement the expression results that pointed towards differences in epithelial to mesenchymal transition (EMT), we evaluated migration and cell attachment. Lastly, in vivo valve development was investigated, as it was an established model of EMT. Results: DAND5-KO EBs progress faster through differentiation. The differences in expression will lead to differences in the expression of genes involved with Notch and Wnt signalling pathways, as well as changes in the expression of genes encoding membrane proteins. Such changes were accompanied by lower migratory rates in DAND5-KO EBs, as well as higher concentrations of focal adhesions. Within valve development, Dand5 is expressed in the myocardium underlying future valve sites, and its depletion compromises correct valve structure. Conclusion: The DAND5 range of action goes beyond early development. Its absence leads to significantly different expression patterns in vitro and defects in EMT and migration. These results have an in vivo translation in mouse heart valve development. Knowledge regarding the influence of DAND5 in EMT and cell transformation allows further understanding of its role in development, or even in some disease contexts, such as congenital heart defects.

Funding: This work was supported by the Fundação para a Ciência e a Tecnologia (PTDC/BIM-MED/3363/2014) and Scientific Employment Stimulus to JI (Norma Transitória 8189/2018), predoctoral fellowship to JG (FCT; PD/BD/136919/2018) and postdoctoral fellowship to FC (DAI/ 2019/08/SAICTPAC/0047/2015), and iNOVA4Health-UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e a Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. Deciphering the clues of a regenerative mechanism for the mammalian adult heart would save millions of lives in the near future. Heart failure due to cardiomyocyte loss is still one of the significant health burdens worldwide. Here, we show the potential of a single molecule, DAND5, in mouse pluripotent stem cell-derived cardiomyocytes specification and proliferation. Dand5 loss-of-function generated the double of cardiac beating foci compared to the wild-type cells. The early formation of cardiac progenitor cells and the increased proliferative capacity of Dand5 KO mESC-derived cardiomyocytes contribute to the observed higher number of derived cardiac cells. Transcriptional profiling sequencing and quantitative RT-PCR assays showed an upregulation of early cardiac gene networks governing cardiomyocyte differentiation, cell cycling, and cardiac regenerative pathways but reduced levels of genes involved in cardiomyocyte maturation. These findings prompt DAND5 as a key driver for the generation and expansion of pluripotent stem cell-derived cardiomyocytes systems with further clinical application purposes. publishersversion published