The Presence of Two MyoD Genes in a Subset of Acanthopterygii Fish Is Associated with a Polyserine Insert in MyoD1.

The MyoD gene was duplicated during the teleost whole genome duplication and, while a second MyoD gene (MyoD2) was subsequently lost from the genomes of some lineages (including zebrafish), many fish lineages (including Alcolapia species) have retained both MyoD paralogues. Here we reveal the expression patterns of the two MyoD genes in Oreochromis (Alcolapia) alcalica using in situ hybridisation. We report our analysis of MyoD1 and MyoD2 protein sequences from 54 teleost species, and show that O. alcalica, along with some other teleosts, include a polyserine repeat between the amino terminal transactivation domains (TAD) and the cysteine-histidine rich region (H/C) in MyoD1. The evolutionary history of MyoD1 and MyoD2 is compared to the presence of this polyserine region using phylogenetics, and its functional relevance is tested using overexpression in a heterologous system to investigate subcellular localisation, stability, and activity of MyoD proteins that include and do not include the polyserine region.

Two closely related ureotelic fish species of the genus Alcolapia express different levels of ammonium transporters in gills.

Most fish excrete their nitrogenous waste across the gills as ammonia through the activity of the Rhesus glycoprotein ammonium transporters. In contrast, fish of the subgenus Alcolapia (Oreochromis) are the only vertebrates that survive the extreme conditions of the soda lakes of Natron and Magadi in East Africa and have evolved adaptations to the highly alkaline waters including the ability to excrete their nitrogenous waste as urea. Nevertheless, Alcolapia retain the Rhesus glycoprotein genes in their genomes and using two heterologous expression systems, we demonstrate that Alcolapia Rhbg is capable of moving ammonia. Comparing ammonia and urea excretion from two closely related Alcolapia species from the same aquarium, we found that while Alcolapia grahami remains fully ureotelic after many generations in lab conditions, Alcolapia alcalica excretes some of its nitrogenous waste as ammonia. Using in situ hybridisation, we demonstrate robust, localised gene expression of Rhbg, rhcg1 and rhcg2 in the gill tissue in both A. alcalica embryos and adults, similar to that in other ammoniotelic fish. In contrast, the expression of these genes in A. grahami gills is much lower than in A. alcalica, suggesting the rapid evolution of a molecular mechanism underlying the complete ureotelism of A. grahami.

Adaptation of the carbamoyl-phosphate synthetase enzyme in an extremophile fish.

Tetrapods and fish have adapted distinct carbamoyl-phosphate synthase (CPS) enzymes to initiate the ornithine urea cycle during the detoxification of nitrogenous wastes. We report evidence that in the ureotelic subgenus of extremophile fish Oreochromis Alcolapia, CPS III has undergone convergent evolution and adapted its substrate affinity to ammonia, which is typical of terrestrial vertebrate CPS I. Unusually, unlike in other vertebrates, the expression of CPS III in Alcolapia is localized to the skeletal muscle and is activated in the myogenic lineage during early embryonic development with expression remaining in mature fish. We propose that adaptation in Alcolapia included both convergent evolution of CPS function to that of terrestrial vertebrates, as well as changes in development mechanisms redirecting CPS III gene expression to the skeletal muscle.

Exploring the Expression of Cardiac Regulators in a Vertebrate Extremophile: The Cichlid Fish Oreochromis (Alcolapia) alcalica.

Although it is widely accepted that the cellular and molecular mechanisms of vertebrate cardiac development are evolutionarily conserved, this is on the basis of data from only a few model organisms suited to laboratory studies. Here, we investigate gene expression during cardiac development in the extremophile, non-model fish species, Oreochromis (Alcolapia) alcalica. We first characterise the early development of O. alcalica and observe extensive vascularisation across the yolk prior to hatching. We further investigate heart development by identifying and cloning O. alcalica orthologues of conserved cardiac transcription factors gata4, tbx5, and mef2c for analysis by in situ hybridisation. Expression of these three key cardiac developmental regulators also reveals other aspects of O. alcalica development, as these genes are expressed in developing blood, limb, eyes, and muscle, as well as the heart. Our data support the notion that O. alcalica is a direct-developing vertebrate that shares the highly conserved molecular regulation of the vertebrate body plan. However, the expression of gata4 in O. alcalica reveals interesting differences in the development of the circulatory system distinct from that of the well-studied zebrafish. Understanding the development of O. alcalica embryos is an important step towards providing a model for future research into the adaptation to extreme conditions; this is particularly relevant given that anthropogenic-driven climate change will likely result in more freshwater organisms being exposed to less favourable conditions.