Abstract
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.
Highlights
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
Human (Homo sapiens), Xenopus tropicalis, and zebrafish (Danio rerio) Transmembrane protein 88 (TMEM88) protein sequences were aligned using Clustal Omega and grouped in an unrooted phyologenetic tree based on sequence conservation
We observed that tmem88aΔ8 mutants had reduced expression of haematopoietic markers, equivalent to the reduction seen by tmem88a morpholino knockdown [9], this was not sufficient to prevent the development of mature primitive erythrocytes and neutrophils as determined by histological staining
Summary
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. 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]. Conditional expression of constitutively active β-catenin, 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]. 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].
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