Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Jonathan Mark Wilson

Advisor Role

Supervisor

Second Advisor

Gabriel Moreno-Hagelsieb

Advisor Role

DAC committee member

Third Advisor

Mathilakath Vijayan

Advisor Role

DAC committee member

Abstract

The gastric proton pump is the key player in gastric acidification, the hallmark of the vertebrate stomach. It is a heterodimeric enzyme encoded by two genes, ATP4A and ATP4B that encode for the α and β subunits, respectively. The acidification of the stomach has been linked to benefits in the increased digestibility of protein and minerals and in the protection against pathogen entry into the intestine. Importantly, an acidic luminal pH is required for the activation of aspartic peptidase pepsinogens to pepsins and the first step of protein digestion. Moreover, studies in diverse vertebrate groups, using a broad range of techniques, have put forward evidence linking gastric acid to increased phosphorus, magnesium and calcium absorption. The role of the gastric proton pump, and, consequently, gastric acid in growth and postprandial energy expenditure has been predicted in fish and other vertebrates through rather indirect procedures of diet modulation or through the use of pharmacological knockdown methods that may introduce off target effects. All in all, there is a lack of an integrative, direct and exhaustive examination of the specific role of the gastric proton pump in animal digestion, energetics and growth. To this end, a proton pump knockout line has been generated through direct targeting of the atp4a gene in Astyanax mexicanus using CRISPR-Cas9 gene editing. Previous Atp4a knockout lines generated in mice lacked an evaluation on the impacts of the proton pump in organismal homeostasis and gut physiology. Moreover, since in teleosts the proton pump is located in the same cell type responsible for the secretion of pepsinogens (the oxynticopeptic cell), it was expected that this knockout would result in a phenotype more closely resembling agastric species (i.e. species that lack a functional stomach with gastric-peptic digestion through evolutionary secondary loss). Indeed, atp4a-nulll A. mexicanus are achlorhydric and present a blunted expression of pepsinogen c (pgc). This knockout line allowed us to analyze transcript levels of candidate genes for apical chloride and potassium movement in the stomach, the identity of which has been a matter of extensive scientific discussion. Specifically, kcne2, kcc4, linked to the apical K+ recycling and the clc2 chloride channel were downregulated in the stomachs of atp4a−/− fish. In addition, through RNAseq analyses we have identified 2064 differentially expressed genes (DEGs) in the stomach and 160 DEGs in the anterior intestine of mutant A. mexicanus. The analyses led to the description of differentially regulated pathways in the stomach and intestine of mutant animals that shed light on the impact of stomach acid in oxidative stress, and lipid and iron metabolism. In particular, we observed a downregulation of pathways associated to oxidative stress, and an upregulation of lipid metabolism transcripts in the stomachs of atp4a-null A. mexicanus. The iron metabolism and transport pathways were downregulated in the intestine of knockout animals. Furthermore, we described a reduction in specific dynamic action (SDA) and in postprandial ammonia excretion, the latter a direct reflection of decreased protein catabolism for energy mobilization in atp4a knockout fish. These changes were not accompanied by a decrease in growth, although carcass analyses showed reduced phosphorus, magnesium, calcium and protein content. The decreased protein levels further highlight the importance of the proton pump to protein digestion and assimilation. Interestingly, this reduction in protein content was accompanied by an increase in total lipid in the body of atp4a−/− A. mexicanus. Remarkably, the knockout animals have developed the capacity to absorb lipid through the gastric mucosa, in contrast to the usual fat absorption restricted to the intestine. Although we have not been able to examine changes in the development of the gut in knockout A. mexicanus, we present a descriptive analysis of the development of the gastrointestinal tract in this species that will open the way to new studies in this emerging model species. We have also compared the phenotypes of heterozygous atp4a+/− and crispants (atp4a/atp4b mutants) to those observed in atp4a−/− A. mexicanus, and our results indicate that for the majority of the traits we have observed, crispant mutants can be used as a rapid, cost-effective alternative to estimate the phenotype of a full knockout. In summary, this thesis presents an integrative analysis of the role of the gastric proton pump and acid-peptic digestion to overall digestion, growth and energy metabolism using direct gene targeting that reduces the confounding effects brought by the use of pharmacological and diet modulation approaches.

Convocation Year

2025

Convocation Season

Spring

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Available for download on Thursday, January 13, 2028

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