Homozygous knockout of the piezo1 gene in the zebrafish is not associated with anemia

Shmukler, Boris E.; Huston, Nicholas C.; Thon, Jonathan N.; Kourkoulis, George; Lawson, Nathan D.; Paw, Barry H.; Alper, Seth L.

doi:10.3324/haematol.2015.132449

Cited by 22 publications

(16 citation statements)

References 18 publications

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“…Our study highlights the possibility that widespread use of antisense knockdown technologies, such as MOs in non-mammalian systems, has contributed to the inconsistencies in NC gene function observed between species. Our findings are also consistent with detailed examples of MOs that give seemingly specific developmental phenotypes, but ultimately do not recapitulate the corresponding genetic mutant (e.g., [18, 19, 37–39]). In addition, a recent study demonstrated that a large majority (~80%) of previously published MO-induced vasculature phenotypes are not observed in the corresponding genetic mutants [16].…”

Section: Discussionsupporting

confidence: 73%

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

2016

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The neural crest (NC) is a stem cell-like embryonic population that is essential for generating and patterning the vertebrate body, including the craniofacial skeleton and peripheral nervous system. Defects in NC development underlie many birth defects and contribute to formation of some of the most malignant cancers in humans, such as melanoma and neuroblastoma. For these reasons, significant research efforts have been expended to identify genes that control NC development, as it is expected to lead to a deeper understanding of the genetic mechanisms controlling vertebrate development and identify new treatments for NC-derived diseases and cancers. However, a number of inconsistencies regarding gene function during NC development have emerged from comparative analyses of gene function between mammalian and non-mammalian systems (chick, frog, zebrafish). This poses a significant barrier to identification of single genes and/or redundant pathways to target in NC diseases. Here, we determine whether technical differences, namely morpholino-based approaches used in non-mammalian systems, could contribute to these discrepancies, by examining the extent to which NC phenotypes in fascin1a (fscn1a) morphant embryos are similar to or different from fscn1a null mutants in zebrafish. Analysis of fscn1a morphants showed that they mimicked early NC phenotypes observed in fscn1a null mutants; however, these embryos also displayed NC migration and derivative phenotypes not observed in null mutants, including accumulation of p53-independent cell death. These data demonstrate that morpholinos can cause seemingly specific NC migration and derivative phenotypes, and thus have likely contributed to the inconsistencies surrounding NC gene function between species. We suggest that comparison of genetic mutants between different species is the most rigorous method for identifying conserved genetic mechanisms controlling NC development and is critical to identify new treatments for NC diseases.

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Section: Discussionsupporting

confidence: 73%

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

2016

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“…Unfortunately, homozygous piezo1b mmr5/ mmr5 embryos did not display any discernible phenotype. A recent report has indicated that KO of the other piezo1 zebrafish paralogue piezo1a also does not produce any discernible phenotype 2 . Although it is possible that zebrafish do not require piezo1 at all for development, based on the drastic consequences observed in mammals and humans when piezo1 is KO or mutated, this seems unlikely 3–5 .…”

Section: Supplemental Informationmentioning

confidence: 99%

Piezo1 is required for outflow tract and aortic valve development

Faucherre

Maati

Nasr

et al. 2019

Preprint

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AbstractAims-During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development.Methods and results-By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. In particular, we have found that mechanical forces generated during the cardiac cycle activate Piezo1 which triggers nitric oxide to be released in the outflow tract. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from a cohort of bicuspid aortic valve patients and identified 3 probands who each harboured a novel variant in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity and defective aortic valve development.Conclusion-These data indicate that the mechanosensitive ion channel piezo1 is required for OFT and aortic valve development and, furthermore, dominant negative variants of PIEZO1 appear to be associated with BAV in humans.

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“…In addition to p53 activation, evidence from Xenopus tropicalis has demonstrated widespread regulation of non-target genes, primarily those with immune function, and mis-splicing of non-target genes in response to morpholino injection (Gentsch et al, 2018). Indeed, there is now mounting evidence, derived primarily from zebrafish, that morphants often do not phenocopy null mutants (Robu et al, 2007;Gerety and Wilkinson, 2011;Kok et al, 2015;Novodvorsky et al, 2015;Rossi et al, 2015;Shmukler et al, 2015;Eve et al, 2017;Joris et al, 2017). In addition, unlike RNAi, where many of the mechanisms of off-target effects have been characterized (Jackson and Linsley, 2004;Seok et al, 2018), the mechanisms of p53 activation, immune responses, splice defects and non-target binding are not as well understood for morpholinos, and minimizing off-target effects may therefore be challenging.…”

Section: Morpholino Antisense Oligonucleotidesmentioning

confidence: 99%

“…Perhaps most worrying is that in a screen of more than 24 genes, 80% of morphant (morpholinotreated; see Glossary) zebrafish did not phenocopy individuals carrying null mutations of the targeted gene, suggesting widespread off-target effects of morpholinos (Kok et al, 2015) and spurring debate in some fields (e.g. Faucherre et al, 2014Faucherre et al, , 2016Shmukler et al, 2015Shmukler et al, , 2016. Here, we provide experimental evidence and present support from the literature to argue that differences between knockdown and knockout-induced phenotypes may not always be a result of non-specific, off-target effects in carefully controlled knockdown experiments.…”

Section: Introductionmentioning

confidence: 99%

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

Zimmer

Pan

Chandrapalan

et al. 2019

Journal of Experimental Biology

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Loss-of-function technologies, such as morpholino-and RNAimediated gene knockdown, and TALEN-and CRISPR/Cas9mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).

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Homozygous knockout of the piezo1 gene in the zebrafish is not associated with anemia

Cited by 22 publications

References 18 publications

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

Piezo1 is required for outflow tract and aortic valve development

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

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