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

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 <i>Astyanax mexicanus</i>. To this end, we generated the first nonmammalian knockout line for the gastric proton pump, <i>atp4a</i> (HKA-α-subunit), in <i>A. mexicanus</i> using CRISPR-Cas9 gene editing. Homozygous mutant <i>atp4a</i>^-/- 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 (<i>pga</i> and <i>pgc</i>) transcript levels were reduced, together with <i>kcne2</i>, <i>kcc4</i> (involved in apical K⁺ recycling), and <i>clcn2</i> (involved in the acid-coupled Cl^- secretion mechanism) mRNA levels. The <i>cftr</i> and <i>slc4a2b</i> transcript levels were significantly increased in knockout stomachs. The gastric morphology and cytology of <i>atp4a</i>^-/- characterized through bright-field and electron microscopy show that the lumen of the gastric glands of <i>atp4a</i>^-/- 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.<b>NEW & NOTEWORTHY</b> We have knocked out stomach acidification in a nonmammalian gnathostome for the first time using CRISPR-Cas9 gene editing in the teleost fish <i>Astyanax mexicanus</i> 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.