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

The human erythrocyte travels nearly 300 miles through 170,000 circuits of the circulatory system during its 120day lifespan. This prolonged voyage subjects the red cell membrane to high and varied shear forces, to compression and stretching when traversing sinusoidal capillary beds, to osmotic shrinkage and swelling when passing through the renal medulla, to oxidative stress during repeated cycles of deoxygenation and re-oxygenation, to assault from the complement system, and to gradual loss of surface area from microvesicle shedding and from macrophage-mediated erythrophagocytosis. Volume regulatory ion transport systems help red cells adapt to these demands, and the transporters and channels that regulate red cell volume are controlled, at least in part, by membrane mechanosensors, including the cation channel, PIEZO1. Gain-of-function mutations in PIEZO1 cause autosomal dominant dehydrated stomatocytosis (DHSt), also known as xerocytosis, characterized by increased cell volume and Na content, decreased K content, and elevated MCHC, with often fully compensated anemia. The PIEZO1 mutants characterized to date in DHSt patients have been associated with delayed inactivation after channel opening. Piezo1 loss-of-function in the mouse is lethal at mid-gestation due to defective vasculogenesis, so the role of PIEZO1 in the mature circulating erythrocyte cannot be studied in the Piezo1 global knockout mouse. Morpholino-knockdown of Piezo1 expression in the zebrafish (Danio rerio) was reported to result in severe anemia, as evidenced by near absence of o-dianisidine staining of 2-day post-fertilization (dpf) embryonic yolk sac and by a 60% reduction in estimated red cell number at 3 dpf. Surviving red cells were noted to be swollen and spherocytic, fragile and dysmorphic. The evidence supporting the specificity of piezo1 knockdown in generation of the anemic phenotype reported by Faucherre et al. was strengthened by reproduction of the observation in three different zebrafish strains using two different piezo1 morpholinos. The effective working doses of morpholino oligomers were titrated to maintain absence of gross developmental defects. These rather high doses injected at the one-cell stage were 8 ng for translation-blocking MO1 (complementary to the piezo1 start codon) and 10 ng for splice-blocking MO2 (complementary to the intron 1/exon 2 splice site). In addition, combined injection of the individually ineffective doses of 4 ng MO1 and 5 ng MO2 decreased embryonic yolk sac hemoglobinization, further supporting a specific effect of piezo1 knockdown. However, recent reports have highlighted substantial differences between the zebrafish phenotypes produced by morpholino oligomer knockdowns and the phenotypes of genomic inactivation of the same genes in as many as 80% of the genes examined. In particular, a morpholino knockdown of piezo1 in zebrafish expressed a phenotype of fin blistering and defective gastrulation that was reasonably interpreted as supporting a role for Piezo1 in maintenance of the ability of epithelial monolayers at steady state to extrude dying cells. However, the fin blistering phenotype was not present in an independent zebrafish line carrying a zinc finger (ZFN)-induced 5 nt deletion in the piezo1 gene, encoding a predicted frameshift in piezo1 exon 8, followed by termination in early exon 9. The erythroid phenotype of that piezo1 strain was not investigated in that work. We have now examined the erythroid phenotype in this zebrafish strain carrying a ZFN genomic knockout of piezo1. Genotyping was performed as previously described. In contrast to the anemic phenotype observed in zebrafish subjected to morpholino knockdown of piezo, the genomic ZFN knockout of piezo1 did not segregate either with anemia in the 3-dpf embryo or with dysmorphic erythrocyte morphology in the adult fish. As shown by o-dianisidine staining of embryonic yolk sac for one clutch of zebrafish progeny arising from the mating of two piezo1 parents (Figure 1), normal hemoglobinization was uniformly evident among 41 embryos in a near Mendelian distribution of piezo1 genotypes among piezo1 (homozygous knockouts, 19.5%), piezo1 (heterozygote knockouts, 65.9%), and piezo1 (WT, 14.6%). cDNA sequencing of pooled embryos confirmed that the ZFN mutant piezo1 allele is transcribed (Figure 2A). The previously reported ZFN-induced genomic lesion indeed produced the expected exon 8 frameshift in the transcribed piezo1 mRNA, encoding the missense frameshift piezo1 mutant polypeptide, DS349L350fs355X. The frame-shifted sequence continues for 7 residues of neo-sequence before encountering an out-of-frame nonsense codon early in exon 9. The presence of this identical mutation was documented by sequencing in multiple individual embryos and adults of piezo1 genotype. To exclude compensatory exon-skipping in the ZFN mutant fish that might have rescued expression of (modified) Piezo1 polypeptide (encoding in its wild-type form a complex