The importance of microbes in animal development: lessons from the squid-vibrio symbiosis

Abstract

Developmental biology is among the many subdisciplines of the life sciences being transformed by our increasing awareness of the role of coevolved microbial symbionts in health and disease. Most symbioses are horizontally acquired, i.e., they begin anew each generation. In such associations, the embryonic period prepares the animal to engage with the coevolved partner(s) with fidelity following birth or hatching. Once interactions are underway, the microbial partners drive maturation of tissues that are either directly associated with or distant from the symbiont populations. Animal alliances often involve complex microbial communities, such as those in the vertebrate gastrointestinal tract. A series of simpler-model systems is providing insight into the basic rules and principles that govern the establishment and maintenance of stable animal-microbe partnerships. This review focuses on what biologists have learned about the developmental trajectory of horizontally acquired symbioses through the study of the binary squid-vibrio model.

Keywords: Euprymna scolopes; Vibrio fischeri; colonization; holobiont; horizontal transmission; morphogenesis.

Figure 1.

The mature symbiosis. (a) A ventral dissection of the adult animal reveals the bilobed light organ in the center of the mantle (body) cavity. (b) A frontal section through one lobe of the organ illustrates the relationship of the three crypts, which house the symbionts, to the surrounding light-modulating tissues; ref, reflector; black box, region of tissue in histological section (c), upper right. (c) Upper, a low magnification view of the tissues shows each V. fischeri (Vf) cell of the symbiont population either in direct contact with host epithelial cells or only a few cell-lengths away; white box, region of the transmission electron micrograph (TEM), lower right. (d) Lower, a high magnification view shows V. fischeri cells (false color, purple) in intimate association with complex, lobate microvilli of the host cell surface.

Figure 2.

Candidate mechanisms underlying molecular and biochemical control of host-symbiont rhythms. (a) escry1 gene regulation. Analyses of the regulation of one of the two host cryptochrome genes over the day/night cycle (environmental light) revealed that the escry1 gene expression peaks during peak luminescence (symbiont light). (b) A mutants defective in light production (Δlux) was unable to induce these symbiosis levels of escry1 expression. This defect could be complemented by exposing symbiotic animals to external blue light (Δlux + light), but not by exposing uncolonized animals (uncolonized + light) (32). (c) (modified from (4)). The host cryptochrome is one of a family of genetic oscillators; it begins to cycle immediately upon colonization by the symbiont. We hypothesize that daily oscillations in the mature organ are governed by both escry1, the genetic oscillator, and peroxiredoxin (PRX), the metabolic oscillator. Whereas in central clocks (the brain), the PRX genes do not cycle, in the peripheral clock (the light organ), expression of these genes does oscillate over the day/night cycle. In addition to the study of gene transcription and protein production, one can characterize these behaviors by analyzing metabolic and ultrastructural features.

Figure 3:

The symbiont’s journey. (a) The anatomy of the host organ - The light organ (dashed circle) as seen through the ventral surface of an anesthetized hatchling animal. A diagram of the external (left) and internal (right) features of this organ; aa, anterior appendage; pa, posterior appendage. (b) Colonization events – Upper, early host (Es, Euprymna scolopes) engagement of the symbiont (Vf, Vibrio fischeri) showing events, critical partner interactions, and biomolecules involved, where known. Left, Confocal image of the organ’s ciliated field, which is shedding abundant mucus in the regions around the pores in response to the MAMPs in environmental seawater; PGN, peptidoglycan (64). Middle, High-magnification confocal image of a living specimen, showing symbiont cells attaching to the cilia (2). Experimental manipulations provide evidence that the host’s perception of symbiont MAMPs, induces both cellular (41) and transcriptomic (43) changes in the host tissues. Right, Colonization in response to a host-generated chitobiose gradient. Following attachment, the symbionts aggregate using an exopolysaccharide (EPS) matrix on their surfaces (90). The observed priming to NO (92) and chitobiose (43) likely occurs in these aggregated cells. Once primed, the V. fischeri cells perceive the host gradient, and chemotax into the organ. Lower, with large numbers of GFP-labeled V. fischeri cells, their transit through tissues can be visualized by confocal microscopy (85); host tissues (red). After entering the pores (Step 1), V. fischeri cells move through ducts (Step 2) lined by outward-beating cilia (bottom TEM), which requires that the symbionts move counter-current. The symbionts then proceed across the antechamber to a bottleneck (Step 3). Once in the crypts, symbiont cells proliferate to fill each crypt by ~12 h post-inoculation (Step 4) (96).

Figure 4.

Symbiont-induced light-organ morphogenesis that follows crypt colonization. (a) The 4-d program of regression of the ciliated epithelial fields on the organ surface occurs through apoptosis, which is induced by symbiont populations deep within the crypts. Left, the process begins with the loss of the ridge of ciliated cells just medial to the pores (Step 1), followed by shortening and loss of the posterior (Step 2) and anterior (Step 3) appendages. (b,c) Changes of the crypt epithelia that result from direct interactions between host and symbiont cells; swelling of crypt epithelial cells (b), and increase in the density of the microvilli on the their surfaces (c). Eliminating V. fischeri cells at 1 or 2 days with antibiotic treatment (dashed lines in graph, (c)) causes a return to the aposymbiotic morphological state of this tissue.