Host evolution shapes gut microbiome composition in Astyanax mexicanus.

The ecological and genetic changes that underlie the evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. Field-collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

Genetic mapping of craniofacial traits in the Mexican tetra reveals loci associated with bite differences between cave and surface fish.

BACKGROUND: The Mexican tetra, Astyanax mexicanus, includes interfertile surface-dwelling and cave-dwelling morphs, enabling powerful studies aimed at uncovering genes involved in the evolution of cave-associated traits. Compared to surface fish, cavefish harbor several extreme traits within their skull, such as a protruding lower jaw, a wider gape, and an increase in tooth number. These features are highly variable between individual cavefish and even across different cavefish populations. RESULTS: To investigate these traits, we created a novel feeding behavior assay wherein bite impressions could be obtained. We determined that fish with an underbite leave larger bite impressions with an increase in the number of tooth marks. Capitalizing on the ability to produce hybrids from surface and cavefish crosses, we investigated genes underlying these segregating orofacial traits by performing Quantitative Trait Loci (QTL) analysis with F2 hybrids. We discovered significant QTL for bite (underbite vs. overbite) that mapped to a single region of the Astyanax genome. Within this genomic region, multiple genes exhibit coding region mutations, some with known roles in bone development. Further, we determined that there is evidence that this genomic region is under natural selection. CONCLUSIONS: This work highlights cavefish as a valuable genetic model for orofacial patterning and will provide insight into the genetic regulators of jaw and tooth development.

The Mexican Tetra, Astyanax mexicanus, as a Model System in Cell and Developmental Biology.

Our understanding of cell and developmental biology has been greatly aided by a focus on a small number of model organisms. However, we are now in an era where techniques to investigate gene function can be applied across phyla, allowing scientists to explore the diversity and flexibility of developmental mechanisms and gain a deeper understanding of life. Researchers comparing the eyeless cave-adapted Mexican tetra, Astyanax mexicanus, with its river-dwelling counterpart are revealing how the development of the eyes, pigment, brain, cranium, blood, and digestive system evolves as animals adapt to new environments. Breakthroughs in our understanding of the genetic and developmental basis of regressive and constructive trait evolution have come from A. mexicanus research. They include understanding the types of mutations that alter traits, which cellular and developmental processes they affect, and how they lead to pleiotropy. We review recent progress in the field and highlight areas for future investigations that include evolution of sex differentiation, neural crest development, and metabolic regulation of embryogenesis.

Genetic mapping of metabolic traits in the blind Mexican cavefish reveals sex-dependent quantitative trait loci associated with cave adaptation.

BACKGROUND: Despite a longstanding interest in understanding how animals adapt to environments with limited nutrients, we have incomplete knowledge of the genetic basis of metabolic evolution. The Mexican tetra, Astyanax mexicanus, is a species of fish that consists of two morphotypes; eyeless cavefish that have adapted to a low-nutrient cave environment, and ancestral river-dwelling surface fish with abundant access to nutrients. Cavefish have evolved altered blood sugar regulation, starvation tolerance, increased fat accumulation, and superior body condition. To investigate the genetic basis of cavefish metabolic evolution we carried out a quantitative trait loci (QTL) analysis in surface/cave F2 hybrids. We genetically mapped seven metabolism-associated traits in hybrids that were challenged with a nutrient restricted diet. RESULTS: We found that female F2 hybrids are bigger than males and have a longer hindgut, bigger liver, and heavier gonad, even after correcting for fish size. Although there is no difference between male and female blood sugar level, we found that high blood sugar is associated with weight gain in females and lower body weight and fat level in males. We identified a significant QTL associated with 24-h-fasting blood glucose level with the same effect in males and females. Differently, we identified sex-independent and sex-dependent QTL associated with fish length, body condition, liver size, hindgut length, and gonad weight. We found that some of the genes within the metabolism QTL display evidence of non-neutral evolution and are likely to be under selection. Furthermore, we report predicted nonsynonymous changes to the cavefish coding sequence of these genes. CONCLUSIONS: Our study reveals previously unappreciated genomic regions associated with blood glucose regulation, body condition, gonad size, and internal organ morphology. In addition, we find an interaction between sex and metabolism-related traits in A. mexicanus. We reveal coding changes in genes that are likely under selection in the low-nutrient cave environment, leading to a better understanding of the genetic basis of metabolic evolution.

Fish models for investigating nutritional regulation of embryonic development.

In recent decades, biologist have focused on the spatiotemporal regulation and function of genes to understand embryogenesis. It is clear that maternal diet impacts fetal development but how nutrients, like lipids and vitamins, modify developmental programs is not completely understood. Fish are useful research organisms for such investigations. Most species of fish produce eggs that develop outside the mother, dependent on a finite amount of yolk to form and grow. The developing embryo is a closed system that can be readily biochemically analyzed, easily visualized, and manipulated to understand the role of nutrients in tissue specification, organogenesis, and growth. Natural variation in yolk composition observed across fish species may be related to unique developmental strategies. In this review, we discuss the reasons that teleost fishes are powerful models to understand nutritional control of development and highlight three species that are particularly valuable for future investigations: the zebrafish, Danio rerio, the African Killifish, Nothobranchius furzeri, and the Mexican tetra, Astyanax mexicanus. This review is a part of a special issue on nutritional, hormonal, and metabolic drivers of development.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

Author Correction: Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic architecture underlying changes in carotenoid accumulation during the evolution of the blind Mexican cavefish, Astyanax mexicanus.

Carotenoids are lipid-soluble yellow to orange pigments produced by plants, bacteria, and fungi. They are consumed by animals and metabolized to produce molecules essential for gene regulation, vision, and pigmentation. Cave animals represent an interesting opportunity to understand how carotenoid utilization evolves. Caves are devoid of light, eliminating primary production of energy through photosynthesis and, therefore, limiting carotenoid availability. Moreover, the selective pressures that favor carotenoid-based traits, like pigmentation and vision, are relaxed. Astyanax mexicanus is a species of fish with multiple river-adapted (surface) and cave-adapted populations (i.e., Tinaja, Pachón, Molino). Cavefish exhibit regressive features, such as loss of eyes and melanin pigment, and constructive traits, like increased sensory neuromasts and starvation resistance. Here, we show that, unlike surface fish, Tinaja and Pachón cavefish accumulate carotenoids in the visceral adipose tissue. Carotenoid accumulation is not observed in Molino cavefish, indicating that it is not an obligatory consequence of eye loss. We used quantitative trait loci mapping and RNA sequencing to investigate genetic changes associated with carotenoid accumulation. Our findings suggest that multiple stages of carotenoid processing may be altered in cavefish, including absorption and transport of lipids, cleavage of carotenoids into unpigmented molecules, and differential development of intestinal cell types involved in carotenoid assimilation. Our study establishes A. mexicanus as a model to study the genetic basis of natural variation in carotenoid accumulation and how it impacts physiology.

Morphogenesis and motility of the Astyanax mexicanus gastrointestinal tract.

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources.

Blueprint for an intestinal villus: Species-specific assembly required.

Efficient absorption of nutrients by the intestine is essential for life. In mammals and birds, convolution of the intestinal surface into finger-like projections called villi is an important adaptation that ensures the massive surface area for nutrient contact that is required to meet metabolic demands. Each villus projection serves as a functional absorptive unit: it is covered by a simple columnar epithelium that is derived from endoderm and contains a mesodermally derived core with supporting vasculature, lacteals, enteric nerves, smooth muscle, fibroblasts, myofibroblasts, and immune cells. In cross section, the consistency of structure in the billions of individual villi of the adult intestine is strikingly beautiful. Villi are generated in fetal life, and work over several decades has revealed that villus morphogenesis requires substantial "crosstalk" between the endodermal and mesodermal tissue components, with soluble signals, cell-cell contacts, and mechanical forces providing specific dialects for sequential conversations that orchestrate villus assembly. A key part of this process is the formation of subepithelial mesenchymal cell clusters that act as signaling hubs, directing overlying epithelial cells to cease proliferation, thereby driving villus emergence and simultaneously determining the location of future stem cell compartments. Interestingly, distinct species-specific differences govern how and when tissue-shaping signals and forces generate mesenchymal clusters and control villus emergence. As the details of villus development become increasingly clear, the emerging picture highlights a sophisticated local self-assembled cascade that underlies the reproducible elaboration of a regularly patterned field of absorptive villus units. This article is categorized under: Vertebrate Organogenesis > From a Tubular Primordium: Non-Branched Comparative Development and Evolution > Organ System Comparisons Between Species Early Embryonic Development > Development to the Basic Body Plan.

Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

Periodic food shortages are a major challenge faced by organisms in natural habitats. Cave-dwelling animals must withstand long periods of nutrient deprivation, as-in the absence of photosynthesis-caves depend on external energy sources such as seasonal floods. Here we show that cave-adapted populations of the Mexican tetra, Astyanax mexicanus, have dysregulated blood glucose homeostasis and are insulin-resistant compared to river-adapted populations. We found that multiple cave populations carry a mutation in the insulin receptor that leads to decreased insulin binding in vitro and contributes to hyperglycaemia. Hybrid fish from surface-cave crosses carrying this mutation weigh more than non-carriers, and zebrafish genetically engineered to carry the mutation have increased body weight and insulin resistance. Higher body weight may be advantageous in caves as a strategy to cope with an infrequent food supply. In humans, the identical mutation in the insulin receptor leads to a severe form of insulin resistance and reduced lifespan. However, cavefish have a similar lifespan to surface fish and do not accumulate the advanced glycation end-products in the blood that are typically associated with the progression of diabetes-associated pathologies. Our findings suggest that diminished insulin signalling is beneficial in a nutrient-limited environment and that cavefish may have acquired compensatory mechanisms that enable them to circumvent the typical negative effects associated with failure to regulate blood glucose levels.

Transorganogenesis and transdifferentiation in C. elegans are dependent on differentiated cell identity.

The differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered "transorganogenesis." We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription factors, but not muscle- or ectoderm- promoting transcription factors, redirects the fate of these organs, suggesting that pharyngeal and somatic gonad cells are specifically competent to adopt intestine identity. Although the intestine, pharynx, and somatic gonad are derived from distant cell lineages, they all express the PHA-4/FoxA transcription factor. While we found that post-embryonic PHA-4 is not necessary for pharynx or uterus reprogramming and PHA-4 is not sufficient in combination with ELT-7 to induce reprogramming in other cells types, knock down of PHA-4 during embryogenesis, which abolishes normal pharynx differentiation, prevents pharyngeal precursors from being reprogrammed into intestine. These results suggest that differentiated cell identity determines susceptibility to transdifferentiation and highlight the importance of cellular context in controlling competency for reprogramming.

Transdifferentiation and remodeling of post-embryonic C. elegans cells by a single transcription factor.

Terminally differentiated post-mitotic cells are generally considered irreversibly developmentally locked, i.e. incapable of being reprogrammed in vivo into entirely different cell types. We found that brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, highly specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult Caenorhabditis elegans. Stable expression of intestine-specific molecular markers parallels loss of markers for the original differentiated pharynx state; hence, there is no apparent requirement for a dedifferentiated intermediate during the transdifferentiation process. Based on high-resolution morphological characteristics, the transdifferentiated cells become remodeled to resemble typical intestinal cells at the level of both the cell surface and internal organelles. Thus, post-mitotic cells, though terminally differentiated, remain plastic to transdifferentiation across germ layer lineage boundaries and can be remodeled to adopt the characteristics of a new cell identity without removal of inhibitory factors. Our findings establish a simple model to investigate how cell context influences forced transdifferentiation of mature cells.

Molecular identification of microorganisms associated with the brine shrimp Artemia franciscana.

BACKGROUND: Prior research on the microorganisms associated with the brine shrimp, Artemia franciscana, has mainly been limited to culture-based identification techniques or feeding studies for aquaculture. Our objective was to identify bacteria and archaea associated with Artemia adults and encysted embryos to understand the role of microbes in the Artemia life cycle and, therefore, their importance in a hypersaline food chain. RESULTS: We used small subunit (SSU) 16S ribosomal RNA gene sequencing to identify bacteria and archaea associated with adults and encysted Artemia embryos from one of their natural environments - Great Salt Lake (GSL), Utah, USA. We found that bacterial sequences most closely related to the genera Halomonas and Vibrio were commonly extracted from GSL adult Artemia, while bacterial sequences most similar to the genera Halomonas, Psychroflexus and Alkalilimnicola dominate in GSL water. Encysted embryos (cysts) yielded bacterial sequences from the genera Idiomarina and Salinivibrio, which were absent from adults and water. Common archaeal sequences in adults were most closely related to the genera Haloterrigena and Haloarcula, while all of the archaeal sequences from GSL water were most similar to the genus Halogeometricum. Cyst derived archaeal sequences were most closely related to the genera Halorubrum and Haloarcula. CONCLUSIONS: In addition to identifying microbial rRNA sequences that are specific to different stages of the Artemia life cycle, we observed striking differences in the sequences associated with the adult Artemia population in samples collected from GSL at different times and locations. While our study was limited in scope and the sample was small, our findings provide a foundation for future research into how the bacteria and archaea associated with Artemia influence the Artemia life cycle, and GSL food web.

Caenorhabditis elegans as a model for stem cell biology.

We review the application of Caenorhabditis elegans as a model system to understand key aspects of stem cell biology. The only bona fide stem cells in C. elegans are those of the germline, which serves as a valuable paradigm for understanding how stem-cell niches influence maintenance and differentiation of stem cells and how somatic differentiation is repressed during germline development. Somatic cells that share stem cell-like characteristics also provide insights into principles in stem-cell biology. The epidermal seam cell lineages lend clues to conserved mechanisms of self-renewal and expansion divisions. Principles of developmental plasticity and reprogramming relevant to stem-cell biology arise from studies of natural transdifferentiation and from analysis of early embryonic progenitors, which undergo a dramatic transition from a pluripotent, reprogrammable condition to a state of committed differentiation. The relevance of these developmental processes to our understanding of stem-cell biology in other organisms is discussed.