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Reply to: Zebrafish prrx1a mutants have normal hearts

Nature volume 585, pages E17–E19 (2020)Cite this article

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The Original Article was published on 23 September 2020

replying to F. Tessadori et al. Nature https://doi.org/10.1038/s41586-020-2674-1 (2020)

To further examine the specificity of the heart phenotype, we generated prrx1a-crispant embryos (G0) with an additional set of guides (Fig. 1a), and using a Cas9 protein optimized to prevent off-target function6,7, in conditions that have been shown to generate mutagenized G0 embryos that lack confounding non-specific traits6. Prrx1a protein cannot be detected in these embryos, confirming the efficiency of the guides (Fig. 1b) and showing that mutations are induced in virtually all copies of the targeted gene in the zebrafish G0 crispant embryos6. These embryos show the same defects that we described previously1—namely mesocardia (although at a lower penetrance) and a reduction in the size of the atrium (Fig. 1c, d). Other defects, such as a smaller head, were also previously observed in prrx1a and prrx1b double mutants8. Notably, both the cilia in the LRO and the expression of spaw appear normal (Fig. 1e, f), indicating that the crispant embryos do not have the early defects in the LRO that were observed in the morpholino-induced mutant embryos and that could non-specifically influence heart laterality. Moreover, the decision of the posterior pole of the heart to move from the midline to the left occurs late—independent of the formation of the LRO and heart jogging9,10. This is also in agreement with our photoablation experiments that were performed after jogging1.

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Fig. 1: prrx1a-crispant embryos show mesocardia and a smaller atrium without early defects in the LRO.
Fig. 2: EMT transcription factors in heart laterality in zebrafish.

Data availability

All of the raw data that support the findings of this study are available within the manuscript and its associated files. Source data are provided with this paper.

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Acknowledgements

Work in the laboratory is supported by grants from the Ministry of Science and Innovation through the Spanish State Research Agency (AEI) (MICIU RTI2018-096501-B-I00) and from Generalitat Valenciana (PROMETEOII/2017/150). M.A.N. also acknowledges the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (SEV-2017-0273) to Instituto de Neurociencias.

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Author notes

  1. Oscar H. Ocaña

    Present address: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

  2. Luciano Rago

    Present address: Department of Oncogenomics, Academic Medical Center, Amsterdam, The Netherlands

  3. Hakan Coskun

    Present address: Boston Children’s Hospital, Harvard Medical School, Harvard University, Boston, MA, USA

  4. Aida Arcas

    Present address: Centro de Investigación Médica Aplicada (CIMA), University of Navarra, Pamplona, Spain

Authors and Affiliations

  1. Instituto de Neurociencias (CSIC-UMH), Sant Joan d’Alacant, Alicante, Spain

    Noemi Castroviejo, Oscar H. Ocaña, Luciano Rago, Hakan Coskun, Aida Arcas, Joan Galcerán & M. Angela Nieto

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  1. Noemi Castroviejo
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Contributions

N.C. performed the majority of the experiments, analysed the data and prepared the figures; L.R. performed CRISPR injections and collaborated on the scoring of heart positioning; and A.A. performed bioinformatics analyses. N.C., L.R. and A.A. did not contribute to the original publication but they contributed to this Matters Arising, mainly because O.H.O. and H.C., the main authors of the previous study, moved to the National Centre for Cardiovascular Diseases (CNIC, Spain) and Harvard University, respectively. C. Minguillón, P. Murawala, E. M. Tanaka and R. Muñoz-Chápuli, the other co-authors of the previous study1, did not contribute to the issues raised in this Reply. They have been informed, and agreed. J.G. designed the CRISPR guides and performed the mutant selection. M.A.N. conceived the study, interpreted the data and wrote the manuscript.

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Correspondence to M. Angela Nieto.

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Castroviejo, N., Ocaña, O.H., Rago, L. et al. Reply to: Zebrafish prrx1a mutants have normal hearts. Nature 585, E17–E19 (2020). https://doi.org/10.1038/s41586-020-2675-0

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