Generation of gastric proton pump atp4a knockouts in Astyanax mexicanus: a fish model for insights into the mechanisms of acidification by oxynticopeptic cells

Abstract

The gastric proton pump H⁺/K⁺-ATPase (HKA) is the highly conserved acid secretory mechanism of the gnathostome stomach. HKA is a heterodimeric pump composed of α and β-subunits. In this study, we have explored the involvement of this enzyme in the transcriptional regulation of pathways linked to acid secretion (Cl^- and K⁺ movement across the cell membrane) and peptic digestion (pepsinogens) in the stomach of the teleost Astyanax mexicanus. To this end, we generated the first nonmammalian knockout line for the gastric proton pump, atp4a (HKA-α-subunit), in A. mexicanus using CRISPR-Cas9 gene editing. Homozygous mutant atp4a^-/- fish appeared healthy but were achlorhydric. The transcript and protein levels of the HKA-β-subunit remained unaltered despite the absence of α-subunit protein. Pepsinogen (pga and pgc) transcript levels were reduced, together with kcne2, kcc4 (involved in apical K⁺ recycling), and clcn2 (involved in the acid-coupled Cl^- secretion mechanism) mRNA levels. The cftr and slc4a2b transcript levels were significantly increased in knockout stomachs. The gastric morphology and cytology of atp4a^-/- characterized through bright-field and electron microscopy show that the lumen of the gastric glands of atp4a^-/- fish was dilated and the oxynticopeptic cells had large cytoplasmic inclusions that were absent in wild-type animals. The tubulovesicular system of knockouts was less developed relative to wild-type animals. Our findings provide novel evidence of the highly conservative nature of the gastric acid-peptic pathways across diverse vertebrates. Furthermore, this work highlights the potential for the use of nontraditional models in biomedical research.NEW & NOTEWORTHY We have knocked out stomach acidification in a nonmammalian gnathostome for the first time using CRISPR-Cas9 gene editing in the teleost fish Astyanax mexicanus targeting the gastric proton pump. This offers a novel and insightful alternative to murine models, having larger offspring numbers, rapid development, and ease of maintenance. In accordance, we present the first demonstration in a knockout animal of how diverse chloride and potassium transporters dynamically respond to-and are directly altered by-acidification.

Keywords: CRISPR-Cas9; H+/K+-ATPase; pepsinogen; tubulovesicular system.