Changing hydrozoan bauplans by silencing Hox-like genes

Abstract

Regulatory genes of the Antp class have been a major factor for the invention and radiation of animal bauplans. One of the most diverse animal phyla are the Cnidaria, which are close to the root of metazoan life and which often appear in two distinct generations and a remarkable variety of body forms. Hox-like genes have been known to be involved in axial patterning in the Cnidaria and have been suspected to play roles in the genetic control of many of the observed bauplan changes. Unfortunately RNAi mediated gene silencing studies have not been satisfactory for marine invertebrate organisms thus far. No direct evidence supporting Hox-like gene induced bauplan changes in cnidarians have been documented as of yet. Herein, we report a protocol for RNAi transfection of marine invertebrates and demonstrate that knock downs of Hox-like genes in Cnidaria create substantial bauplan alterations, including the formation of multiple oral poles ("heads") by Cnox-2 and Cnox-3 inhibition, deformation of the main body axis by Cnox-5 inhibition and duplication of tentacles by Cnox-1 inhibition. All phenotypes observed in the course of the RNAi studies were identical to those obtained by morpholino antisense oligo experiments and are reminiscent of macroevolutionary bauplan changes. The reported protocol will allow routine RNAi studies in marine invertebrates to be established.

Figure 1. Observed phenotypic changes in knock down experiments of Hox-like genes in the hydrozoan Eleutheria dichotoma.

Four of the five Hox-like genes produced phenotypically abnormal medusae (PAMs) in gene knock down studies. The main features of bauplan change relate to additional tentacle bifurcation (Cnox-1; see arrows), multiple oral poles or heads (Cnox-2), head duplication (Cnox-3; see arrows), and oral-aboral body axis deformation (Cnox-5). The two upper panels show life pictures of medusae transfected with double stranded RNA (B_1,2, C_1,2, D_1,2, E_1,2), and the lower panel medusae transfected with morpholino oligos (B_3,4, C_3,4, D_3,4, E_3,4). A_1,2 and A_3,4 are controls, scale bar is 100 µm. While we here unambiguously show that Hox-like genes can be silenced we think it would be premature to derive final conclusions on their functions yet.

Figure 2. Time course for the development of phenotypic effects resulting from Cnox-5 gene knock down.

Both morpholino oligo and RNAi experiments result in an increase of phenotypically abnormal medusae (PAMs) over time. Shown is the time course of increasing numbers of PAMs as a result of inhibition by double stranded RNA and inhibition by morpholino-antisense oligonucleotides. In each case control 1 is untreated animals cultured in normal seawater, and control 2 is a sense morpholino oligonucleotide or dsRNA targeted to the Trox-2 gene from the placozoan Trichoplax adhaerens respectively. Independent controls were run for each single experiment.

Figure 3. In situ expression of Hox-like genes in the hydrozoan Eleutheria dichotoma.

The five Hox-like genes, Cnox-1 to Cnox-5, display differential spatio-temporal expression patterns along the oral-aboral body axis. Cnox-1 (A₁–A₄) is expressed ectodermally in the so-called “Nesselring”, an area of undifferentiated cells lining the ring canal of medusae (cross section: A₃, A₄). Cnox-2 is expressed in the entoderm of developing medusa buds (B₁, B₂), in 2-day-old planula larvae ectodermally and orally (B₅), in 5-day-old planula larvae along the body column ectodermally (B₇) and at the oral pole of primary (B₉) and adult polyps (B₃; single polyp with 4 tentacles). Cnox-4 is exclusively expressed at the aboral pole of primary polyps (C₂). Cnox-3 expression perfectly marks the most ectodermal oral part of the manubrium (D₁, D₂). Cnox-5 shows a remarkable pattern of expression moving from aboral only in the planula larva (E₂) to both oral and aboral simultaneously in the metamorphosing polyp (E₄). NBT/X-phosphate (A₁, A₄, B₁–B₃, D₁) and fluorescein-labeled probes (A₂, B₅, B₇, B₉, C₂, D₂, E₂, E₄). Signals in B₅, B₇, B₉, C₂, E₂ and E₄ are overlaid with DAPI staining. Morphologies are shown in light microscopy (B₄, B₆, B₈, C₁, E₁, E₃). Scale bar is 50µm. A₂, D₁, E₁–E₄ are reprinted with permission from Elsevier Publishers .