Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes

RESEARCH ARTICLE Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes Alexander M. J. Eve, Elsie S. Place, James C. Smith* Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom * Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Eve AMJ, Place ES, Smith JC (2017) Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes. PLoS ONE 12 (2): e0172227. doi:10.1371/journal.pone.0172227 Editor: Yann Gibert, Deakin School of Medicine, AUSTRALIA Received: July 1, 2016 Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos. Accepted: February 1, 2017 Published: February 13, 2017 Copyright: © 2017 Eve et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Francis Crick Institute (www.crick.ac.uk), which receives its core funding from Cancer Research UK (FC001157), the UK Medical Research Council (FC001157), and the Wellcome Trust (FC001-157). We are also grateful to the Fondation Leducq (www. fondationleducq.org) for a Transatlantic Network of Excellence Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction The requirement for Wnt signalling in haemovascular development in vertebrates is complex, with activation of the Wnt pathway capable of both promoting and of inhibiting haematopoiesis. Early in vitro studies showed that overexpression of β-catenin increased proliferation of haematopoietic stem cells (HSCs), whereas use of Wnt inhibitors prevented HSC growth and reduced their ability to reconstitute the haematopoietic system when transplanted into irradiated mice [1]. Subsequently, conditional expression of constitutively active β-catenin, specifically in HSCs, caused a transient expansion of the HSC pool, but it did so at the expense of self-renewal and differentiation, resulting in blood cell depletion and death [2]. Furthermore, HSCs expressing a stable form of β-catenin failed to develop into downstream erythromyeloid lineages and they lost repopulation activity [3]. In vitro analyses of mouse HSCs with different hypomorphic mutations in Apc show that these cells have increased Wnt levels, increased rates of differentiation, and reduced proliferation [4]. Similar results were obtained in Wnt3a -/- mice, where HSCs were significantly fewer in number, with poor self-renewal and repopulation potential [5]. In contrast, the non- PLOS ONE | DOI:10.1371/journal.pone.0172227 February 13, 2017 1 / 17 Comparing tmem88a mutants and morphants Competing interests: The authors have declared that no competing interests exist. canonical Wnt ligand Wnt5a enhances self-renewal and promotes quiescence of HSCs, and this is thought to occur by interfering with the ability of Wnt3a to activate the canonical pathway [6]. Indeed, this might be conserved in mammals because human HSCs transplanted into irradiated mice had a greater reconstitution capacity when treated with Wnt-5a conditioned medium [7]. Together, these data suggest that dynamic regulation of canonical and non-canonical Wnt signalling is necessary to balance both blood cell expansion and differentiation. However, we note that many of these studies make use of exogenous activation of the Wnt pathway and the way in which Wnt signalling is modulated during haematopoiesis in vivo is poorly understood. Transmembrane protein 88 (TMEM88; ENSG00000167874) is a two-transmembrane protein with a valine-tryptophan-valine (VWV) motif at the C-terminus that binds the PDZ domain of dishevelled (Dvl). In HEK293 cells, RNAi knockdown of TMEM88 increased Wnt activity, while overexpression of TMEM88 attenuated Wnt1-induced activation. A Siamois reporter in Xenopus showed that TMEM88 inhibits Wnt activation by xDvl, also suggesting that TMEM88 negatively regulates Wnt signalling [8]. The zebrafish orthologue, tmem88a (ENSDARG00000056920), is expressed in the heart fields, vasculature, and blood islands. This suggests that it might regulate Wnt signalling in these tissues and thereby influence their development in vivo [9,10]. Three studies have explored this question. Our own work has shown that tmem88a is enriched in fli1a:gfp+ve endothelial cells between 26–28 hpf, and morpholino oligonucleotide (MO) knockdown of tmem88a inhibited primitive blood development at 48 hpf [9]. Novikov and Evans showed that tmem88a is expressed downstream of gata5/6 in the heart field but is expressed independently of gata5/6 in the posterior blood island. MO knockdown of tmem88a reduced expression of nkx2.5 in the heart field at the 8-somite stage (8ss), reduced the expression of cardiomyocyte markers at the 23-somite stage, and reduced the number of cardiomyocytes in Tg(myl7:DsRed2-nuc) embryos at 48 hpf. Heat-shock inducible expression of the Wnt antagonist dikkopf 1 (dkk1) was sufficient to rescue expression of nkx2.5 and to restore the number of myl7:DsRed2+ve cardiomyocytes. In addition, overexpression of fulllength tmem88a reduced nkx2.5 expression in cardiac progenitors and this expression was rescued by heat-shock inducible wnt8 expression. This study also observed a significant reduction in gata1 and spi1 expression in embryos at the 8-somite stage, further suggesting a requirement for tmem88a in primitive haematopoiesis [10]. Most recently, Musso and colleagues showed that the hearts of Tmem88a-deficient zebrafish embryos had increased ventricular conduction velocity at 48 hpf, although the decrease in the number of ventricular nuclei reported by Novikov and (...truncated)