Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis

Abstract

The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization.

FIG 1

Anatomy of the E. scolopes light organ. (A) An anesthetized animal was imaged using a dissection scope, revealing the light organ and ink sac (dashed box) through the translucent mantle tissue. (B) The host light organ counterstained using acridine orange as a live stain. The light organ is visible above the ink sac, and two pairs of ciliated appendages splayed out to each side can be seen. The light organ is bilobed, with a pair of appendages, three pores, and three crypts being present on each side. (C) Model of the colonization area and process. The red dashed line indicates the general path that V. fischeri cells must follow as they pass from the ambient seawater to the interior of the host's light organ. Once they are inside the mantle cavity, the bacteria attach to the ciliated field (cf) before migrating to the pores (p) at the base of the light organ appendages. They then pass into the ducts (d) and through the antechamber (a) before pausing at the bottleneck (b). Only one or a few bacteria pass the bottleneck before it constricts, and once they are past the bottleneck, they enter the deep crypts (c), where they are able to multiply and begin to luminesce.

FIG 2

Visualization of multiple transcripts within animal tissues. Representative images at ×10 (A) and ×40 (B) magnifications of the EsHsp90 transcript (blue) and the actin transcript (green) are shown. (A) The actin transcript is present throughout much of the light organ and strongly expressed in striated muscle (arrowhead). (B) In the enlarged area encompassed by the dashed box, the actin transcript is not detectable either in the ciliated field (cf) around the pore (p) or in the epithelium of the appendages, although it is easily observed in the vascular tissue within the appendage (arrowhead). In contrast to the actin transcript, the probe for the EsHsp90 transcript was detected strongly and uniformly throughout the appendages and ciliated field. Ant., anterior; Pos., posterior.

FIG 3

Tracking the bacterial position during initiation of colonization. For all images, bacteria were labeled with probes specific for 16S rRNA (green), and squid tissue was stained with Alex Fluor 633-phalloidin (blue). (A′ to D′) Enlargements of the boxed regions in panels A to D, respectively. Magnifications, ×40. (A, A′) Before exposure, no bacteria were visible within the light organ of the host. (B, B′) After 3 h of exposure, bacteria (arrowheads) have associated with the host and begin to be visible within the light organ ducts, located immediately in the interior of the pores (Fig. 1C). (C, C′) By 6 h postexposure, bacteria have migrated into the light organ and have begun to colonize the crypt space. (D, D′) After 24 h of exposure, the host is bioluminescent and the symbionts are visible throughout the crypts.

FIG 4

Tracking expression of luxA during host colonization. (A and B) Visualization of V. fischeri by labeling of the 16S rRNA (green); (A′ and B′) expression of the luxA gene (red); (A″ and B″) visualization of host tissue by labeling the EsHsp90 transcript (blue), in which the label is overlaid with the two bacterial labels; (A, A′, and A″) at 6 h after inoculation, V. fischeri (arrows) has migrated into the crypts but has not yet grown to a sufficient density to induce strong expression of the luxA gene; (B, B′, and B″) after 24 h, when the symbionts (arrows) are densely packed and brightly bioluminescent, V. fischeri shows strong expression of the luxA gene.

FIG 5

The EsPgrp1/2 transcript level is elevated predominantly in the crypts by 24 h postcolonization. (A and B) Visualization of V. fischeri by labeling of the 16S rRNA (green); (A′ and B′) expression of EsPgrp1/2 (red); (A″ and B″) visualization of host tissue by labeling the EsHsp90 transcript (blue), in which the label is overlaid with the two other labels; (A, A′, and A″) EsPgrp1/2 is expressed in the symbiotic (Sym.) crypt epithelium at 24 h postcolonization; (B, B′, and B″) in contrast, its expression is undetectable in the crypts of aposymbiotic (Apo.) animals at the same stage of development. d, duct; a, antechamber; b, bottleneck; c, crypt (as shown in Fig. 1).

FIG 6

Tracking of a host transcriptional response to the bacterial symbiont. The light organs from aposymbiotic (A) and symbiotic (B) animals at 24 h postcolonization were labeled with three probes. EsHsp90 (blue) labels the light organ throughout, but EsCadDP1 (red) is found only in the ducts (d) and antechamber (a) adjacent to the V. fischeri (whose 16S rRNA is labeled green)-colonized crypts (c). (C) The EsCadDP1 signal is localized to the ducts and not the epithelium in contact with the bacteria. Ant., anterior; Pos., posterior; cf, ciliated field (as in Fig. 1). Magnifications, ×10 (A and B) and ×40 (C).