Right on cue: Microbiota promote plasticity of zebrafish digestive tract

Michelle S. Massaquoi and Karen J. Guillemin


All animals on earth have evolved within a world teeming with microscopic life. The genesis of bacteria dates back 2.6 billion years, whereas primitive humans evolved only 315 thousand years ago from ancestors who had always coexisted with microbes. We refer to the community of bacteria, viruses, fungi, and archaea inhabiting a multicellular host as its microbiota. Only recently have biologists considered an organism as an ecosystem of many, rather than an isolated individual. Although microbes were initially vilified as pathogens, just a small fraction of the thousands of microbial species cause disease in animals. Microbial life is now being appreciated for its multitude of roles in host homeostasis. As our knowledge of the intricate interactions between host organisms and resident microbiota increases, it is fundamentally changing how we view many aspects of animal biology, including animal development.

Viewed from an evolutionary perspective, microbes have shaped animal history by influencing their fitness throughout their lifespans. As described by the Modern Synthesis, natural selection dictates how organisms that are better adapted to their environment will pass down their genes to the next generation. Resident microbes can shift that fitness landscape for mature organisms, for example by supplying degradative enzymatic capacities to allow hosts to access new sources of nutrition. Additionally, microbes can influence which specific genotypes survive and reproduce by shaping the developmental programs that determine how an organism forms from a single-cell embryo to a mature multicellular adult. Polyphenism is a biological phenomenon in which distinct phenotypes can arise from a single clonal genotype, demonstrating the plasticity of developmental biology. Predation, temperature, and nutrient availability are all direct environmental factors that induce polyphenism.

Resident microbes can also be added to this list of factors that influence a host’s developmental trajectories. For example, the presence or absence of the bacterium Wolbachia significantly impacts ovary and oocyte development in the parasitic wasp, Aosbara tabida Nees (Dedeine et al. 2001).

Vertebrates harbor dense and complex microbiota, especially in their digestive tracts (Ley et al. 2008). The impact of the microbiota can be studied through the use of microbiologically sterile, or “germ-free” animals and with gnotobiology experiments, using biological systems in which all members of a community are known. In germ- free mice, not only does the absence of the microbiota impair maturation of the gut and associated mucosal immune system (Belkaid and Harrison 2017), but many distal organ systems are also impaired (Schroeder and Backhed 2016). For example, germ-free mice have stunted development of their intestinal villi capillary network (Stappenbeck et al. 2002) as well as incomplete bone formation (Sjogren et al. 2012). Although the majority of germ-free and gnotobiotic studies of vertebrates have been conducted in laboratory mice, comparisons across other vertebrate models are invaluable for understanding which host responses to microbiota are conserved across multiple host lineages.

In this chapter, we will discuss insights about the developmental impacts of the microbiota in the model vertebrate Dcmio rerio, the zebrafish. George Streisinger at the University of Oregon pioneered the use of zebrafish as a model system for developmental and genetic research (Grunwald and Eisen 2002). There are many advantages to working with zebrafish as a model vertebrate. In addition to genome conservation with mammals, zebrafish are genetically tractable, with many mutant and transgenic lines readily available. They also have high fecundity and are optically transparent during embryonic to larval stages, making this model organism ideal for studying developmental processes in real time. Additionally, because zebrafish develop ex utero, they can easily be derived germ-free by surface sterilization of the outer chorion for experiments aiming to understand host-microbe interactions (Melancon et al. 2017). Culture collections of zebrafish-associated bacterial isolates, with draft genome sequences, are available, enabling gnotobiotic experimentation (Stephens et al. 2016). Together, the transparent properties of larval zebrafish with the use of transgenic lines— that allow tracking of specific cell types and gnotobiological experiments—enable a high-resolution perspective of how the microbiota influence host development. Below, we discuss the ways in which the gut microbiota impact different aspects of zebrafish larval development, following the animals’ first exposure to environmental microbes upon hatching out of their chorions. We use insights gleaned from gnotobiotic zebrafish studies to speculate how host-microbe interactions evolved to modulate developmental program plasticity to optimize the organisms' fitness for different environments.

Development under immune surveillance

The vertebrate intestine serves multiple roles, both as an organ for food digestion and nutrients absorption and as an immunological organ for harboring the body’s most abundant microbial population. The intestinal epithelium can be thought of as the “inner skin” of the body because like the epidermis, it interacts with the outside environment, not only interfacing directly with microbial cells inhabiting the intestinal lumen, but also the rich source of bioactive molecules they secrete (Fischbach and

Segre 2016). With the multitudes of microbial life that populate any given vertebrate, a healthy immune system continuously monitors the intestinal lumen and senses threats posed by resident or invading microbes. Appropriately balanced responses to the microbiota are critical for symbiosis because on the one hand, lack of defense can lead to microbial growth and on the other hand, excessive inflammatory responses can be detrimental to both host and microbe cell populations. As the host develops, the resident microbes help train the immune system to achieve this appropriately balanced response (Belkaid and Harrison 2017).

The gnotobiotic zebrafish model has allowed a detailed characterization of the different immune responses elicited by individual members of the microbiota (Murdoch and Rawls 2019). For example, zebrafish mono-associated with different zebrafish-derived bacterial isolates will exhibit different levels of immune gene expression (Rawls et al. 2006) and accumulate different numbers of neutrophils, a type of white blood cell that lead the immune system’s inflammatory response (Rolig et al. 2015). These types of data inspire the question of how different bacterial residents elicit different immunological responses in the host. Two possible and not mutually exclusive mechanisms are that host immune sensors are differentially stimulated by different bacteria, and that different bacteria produce immunomodulatory factors altering host immune responses.

During larval development, zebrafish rely on their innate immune system for microbial sensing prior to the maturation of their adaptive immune system in juvenile stages. The best characterized of their innate immune sensors are the Toll-like family receptors (TLRs), which are part of an ancient pattern recognition family of receptors (Jault. Pichon, and Chluba 2004; Deguine and Barton 2014). TLR activation is mediated by the sensing of generic microbial products termed microbial-associated molecular patterns (MAMPs), such as cell wall components and flagellin. which subsequently regulates the appropriate immune response (Deguine and Barton 2014). The specificity and downstream response of TLR signaling is partly dictated by the differential recruitment of intracellular adaptor molecules. Myeloid differentiation primary response 88 (MyD88) (Hall et al. 2009) is a common adaptor of TLRs and Interleukin-1 receptor that regulates the expression of pro- or antiinflammatory cytokines and mitogen-activated protein kinase (МАРК) signaling for cell survival or proliferation (Akira 2003; Larsson et al. 2012). The zebrafish genome has duplicated tlr genes with a single copy of myd88, that has conserved function in modulating innate immune responses (Jault et al. 2004; Meijeret al. 2004; Van Der Sar et al. 2006; Bates et al. 2007; Hall et al. 2009; Burns et al. 2017). Of note, Myd88-deficient zebrafish have a completely attenuated intestinal neutrophil influx, indistinguishable from germ-free animals (Bates et al. 2007; Burns et al. 2017). This indicates that much of the immunological responses to the intestinal microbiota are mediated through Myd88. One trait that varies dramatically across zebrafish bacterial isolates is their capacity for motility within the intestine (Schlomann et al. 2018; Wiles et al. 2018). Variation in bacterial in vivo production of motility machinery, such as flagellin subunits, could account for different capacities of different bacterial isolates to activate Myd88-dependent immune responses.

Resident bacteria also modulate host immune responses through specific secreted factors. For example. Rolig and colleagues demonstrated that a Shewanella isolate was an especially potent suppressor of neutrophil intestinal influx, a response that could be recapitulated with Shewanella secreted factors (Rolig et al. 2015). More recently, Rolig and colleagues showed that several Aeromonas strains secrete a protein, Aeromonas immune modulator A (AimA), that dampens neutrophil influx and proinflammatory cytokine expression and also confers a colonization advantage to the bacteria (Rolig et al. 2018). The crystal structure of AimA revealed that it has two distinct domains with related folds, both individually retaining the capacity to regulate neutrophils. To investigate whether AimA confers a colonization advantage to Aeromonas through attenuating inflammation, the authors measured the abundance of Aeromonas strains w'ith and without AimA in myd88 mutant zebrafish. They found that in these immunocompromised hosts with a limited immune response, Aeromonas no longer required AimA for maximal intestinal colonization, suggesting that in the face of a normal host immune response, the bacteria benefit from AimA’s ability to dampen inflammation (Rolig et al. 2018). This study illustrates that Myd88- mediated host responses to the microbiota modulate features of the host environment that impact the fitness of resident bacteria. This finding is corroborated by the fact that isolates of Aeromonas experimentally evolved to colonize wild type zebrafish intestines are less fit when introduced into myd88 deficient hosts (Robinson et al. 2018). Thus, the innate immune system acts as a conduit to intercept and respond to microbial cues and may underlie microbiota-mediated developmental plasticity.

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