Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri

Abstract

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.

Keywords: allorecognition; ascidian; gene expression; stem cells; tyramide signal amplification.

Figure 1. Flowchart of single and double whole-mount fluorescent in situ hybridization (FISH) protocols

Times listed next to arrows are the length of time needed to complete the step described in the preceding text box. A single-color FISH experiment requires approximately four days to complete, and a two-color FISH experiment requires approximately one day more.

Figure 2. Expression patterns of markers for developing and adult structures in B. schlosseri

(a-b) atp1a3 is expressed by cells of the pyloric caecum in the digestive system of the adult zooid (a) and the primary bud at stage C1 (b, arrow). atp1a3 expression is also present in part of the developing endostyle and adjacent tissues in primary buds at stage C1 (b, arrowheads indicate expression visible on the left side of the endostyle). (c) vwfl2 expression is present in the ventral portion of the endostyle of the zooid. (d) atp1a3 (red, arrow) and vwfl2 (green) can be simultaneously visualized in adult zooids by two-color FISH. atp1a3 expression is also visible in individual non-epithelial cells of unknown identity that are associated with the adult zooid. Scale bars indicate 50 μm.

Figure 3. Expression patterns of markers for developing structures in B. schlosseri

(a) fgf11-14 is expressed by a subset of cells in the developing cerebral ganglion (dashed line) of the primary bud at stage C1. (b-c) par6 expression is present in epithelial cells of the primary (b) and secondary (b and c) buds. Primary and secondary buds in panel b and secondary bud in panel c are demarcated by dashed lines. (d-f) runx is expressed in a stage-specific manner. At stage A2, expression of runx is present throughout the secondary bud (d, dashed lines). At stage B1, runx expression is restricted to the boundary between the primary and secondary buds (e, dashed lines). By stage C1, runx is no longer expressed by cells of the secondary bud (f). Faint staining inside the secondary buds at stages A2, B1, and C1 is indicative of trapping of reagents, and is present in a region devoid of cells (d-f). Background staining is also evident outside of the demarcated areas in panels d-f as a result of non-specific binding of reagents to the tunic and the surface of the primary and secondary buds. Solid lines in panels e and f define the portions of the secondary bud where runx expression is not detected. Scale bars indicate 50 μm.

Figure 4. Expression pattern of marker for mitotically active cells in B. schlosseri

(a-c) h3 is expressed by a portion of the cells in the primary and secondary bud at stages A1 (a) and A2 (b). The majority of cells in the secondary bud at stage A2 strongly express h3 (c). Primary buds in panels a and b and the secondary bud in panel c are demarcated by dashed lines. (d) h3 expression is also present in clusters of blood cells known as “cell islands” on the ventral side of the zooid at stage C1 (surrounded by dashed lines) on both sides of the endostyle (marked by arrowheads). Other non-epithelial cells of unknown identity outside of these clusters also exhibit h3 expression. The primary and secondary buds in panel d are demarcated by solid lines. Scale bars indicate 50 μm.

Figure 5. Expression patterns of markers for germline and germline-associated cells in B. schlosseri

(a) vasa is expressed by clusters of germ cells associated with the secondary bud (dashed line) and just posterior to the secondary bud at stage B2. (b) At stage B1, piwi-positive germ cells are present posterior to the secondary buds (dashed lines). (c-d) tgfβ-f expression is detected in clusters of primitive follicle cells posterior to the secondary bud (dashed lines) at stage A2 in non-fertile juveniles (c) and in follicle cells surrounding the maturing oocytes (o) and testes (t) in the primary buds of fertile animals (d). (e-f) Two-color FISH can be used to simultaneously visualize vasa expression (red) and tgfβ-f expression (green) in non-fertile (e) and fertile (f) animals. Scale bars indicate 50 μm.