Eye-Specification Gene Expression in E. Scolopes Light Organ

The Hawaiian bobtail squid, Euprymna scolopes, is a common model organism for studying neurons and eye development. One unique feature of the squid that is particularly interesting for studying development is the light organ.

Hawaiian Bobtail Squid

Hawaiian Bobtail Squid [3]

Vibro Fischeri Micrograph (fluorescently stained)

Vibro Fischeri Micrograph (fluorescently stained). [4]

The bobtail squid enters into a mutualistic relationship with the bacteria Vibrio fischeri. This mutualistic relationship is established in the light organ, located by the ink sac on the mantle. From the mutualism, the bacteria get an environment to colonize, and the squid is able to use the bacteria to produce light. The Vibro fischeri use quorum sensing molecules to regulate their gene expression, notably the expression of the Lux protein, which bioluminesces. The squid are able to use the light organ and light it produces to counter-illuminate themselves so there is no shadow for predators to see when they are swimming at night (Visik and Ruby, 2006).

There are two particularly interesting stages in development of the light organ: genealogical development and its eventual colonization by the bacteria. The development, as shown by Peyer et. al below, shows similarities to the squid’s eye development. Additionally, the bacterial colonization of the light organ is unique to the Vibro fischeri. As described in Visik and Ruby, all squids are born without any bacteria in their light organ. Within 12 hours after hatching, the bacteria have migrated through the squids mucus into its pores and are making their way into the three crypts deep inside the light organ. Only V. fischeri make it to colonize the light organ, despite the fact that the squid pulls these bacteria out of the water column which is littered with countless other bacterial species. By the time the bacteria reach 10^6 population densities, they induce bioluminescence. At the end of every day, the squid ejects 90-95% of the bacteria from the light organ, and during the day populations grow to 10^6 again by the night (Visick and Ruby, 2006).

The Paper

Peyer et al. noticed in their paper “Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues”, a similarity between the tissues in the squid’s eyes and its light organ. They point out that despite the coevolution of eyes in the animal kingdom, many genes are homologous in eye development such as pax6, eya, dac, and six. These genes code for transcription factors that up regulate eye morphogenesis and are often called the “eye specification” genes. Similarities in E. scolopes between the eyes and light organ include a lens with crystilin proteins, a reflective layer of proteins on the bottom with homology to reflectin proteins, and proteins involved in phototransduction (light getting converted into electrical neurological signals).

The light organ and eyes are not homologous, as the eye develops from the ectoderm and the light organ develops from the mesoderm. They also develop at different stages in development. Final light organ development is dependent on the colonization of the V. fischeri, which induces morphological changes in the light organ such as apoptosis of light organ appendages and ciliated ridges. In the paper, Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes and their expression in response to symbiont cues, Peyer et al. asked the question whether eye specific genes were expressed in the light organ, and aimed to analyze their response to the symbiosis with the V. fischeri.

 

Methods

To test gene expression patterns, Peyer et al. tested light organ tissues for full length cDNA using primers of the eye genes pax6, eya, dac, and six. They also examined all of these genes globally using a full-mount in situ hybridization during embryogenesis and postembryonic development. For the postembryonic work, they divided the developed squid into 4 test groups: newly hatched, uncolonized, colonized by WT V. fischeri, and colonized by mutant V. fischeri. The mutants had the Lux protein deleted, preventing them from luminescing. They then compared expression data between the differentially colonized squid to see if colonization or the bioluminescence of the bacteria caused gene expression changes. Real time PCR was also used to see transcripts of the pax6, eya, dac, and six.

Results

Peyer et al. found that all four genes were expressed both in the eye and light organ, as is evident in Figure 2 below, showing embryo in-situ hybridization sampled at progressive stages of development, S-18, S-22, and S-26. Pay close attention to the letters “e” for eye, and the white box which symbolizes the light organ although it is below the mantle and difficult to see in some pictures.

Figure 2

Figure 2: whole-mount embryo in situ hybridization showing eye gene expression both in the light organ and in the eyes. (Peyer 2014) Tissues showing transcript signal include
the eye (e), optic lobe (ol), tentacles (t), statocysts (s), shell gland (sg), mantle (m), funnel fold (f), gill (g), fin (f), nuchal organ (n), optic nerve (on), and olfactory organ (o).

Further, the four gene transcripts are localized in the light organ, as is evident in the whole mount in situ hybridization, Figure 3 below.

Figure 3

Figure 3: whole-mount embryo in situ hybridization showing the light organ specifically. This shows clearly that the eye specific genes play a role in light organ development (Peyer 2014). Areas of interest highlighted include (is) showing the Ink Sack and (hg) showing the hind gut.

Symbiotic effects had drastic changes to some gene expression of the eye specific genes, especially in the surface tissues of the light organ. However, pax6 genes had no significant change between WT and lux mutant bacteria. eya expression was similar, only significant change for the anterior and posterior appendages when comparing colonized to uncolonized squid. Colonized squid did not have any significant eya expression difference between WT and lux mutant bacteria.

In contrast, six transcript was significantly above background with WT but not for lux mutant bacteria. six expression was not significantly different between lux mutants and uncolonized squid. This shows an effect of the light production by v. fischeri on six expression. 

For dac expression, similar to six, WT colonized light organs were significantly above background levels but lux mutants were not. This trend was only significant on the pores though. All of the above transcripts and their differential expression are evident in Figure 4 below.

Figure 4

Figure 4: Expression of the pax6, eya, six, and dac gene transcripts in surface tissues of light organs by whole-mount in situ hybridization (Peyer 2014). Areas of interest include Light-organ tissues such as anterior appendages (grey arrows), posterior appendages (brown arrows), ciliated ridges (blue arrows), and pores (green arrows).

The RT-PCR analysis showed that pax6, eya, and six transcripts did not differ significantly between colonization conditions. However, the dac transcript differed significantly over the entire light organ, and was significantly up regulated in light organs colonized by lux mutant V. fischeri. See Figure 8 below.

Figure 8: Gene expression in differentially colonized light organs

Figure 8: Gene expression in differentially colonized light organs (Peyer 2014). Hatchling is an uncolonized recently hatched squid. Apo is 24 hour juvenile squid with no colonization. Sym is a wild type colonized squid, deltaLux is a squid colonized by lux knockout V. fischeri.

Discussion

The study proves that eye specific genes are expressed in both the eye and the light organ, and that transcripts did depend on the cues or presence of the symbiotic bacteria. Additionally, the morphological changes due to the colonization of the light organ are produced by such cues or presence, including the coalescence from three pores on the uncolonized light organ a single pore used by the adults to excrete 90-95% of the bacteria every night. These morphological changes either do not happen or happen late in uncolonized squids and in lux mutant squids. They go on to say that Notch signaling often regulates Pax6 which auto regulates and regulates eya, six, and doc genes. They theorize that perhaps Notch pathways could be leading to the differential expression of Pax6 and other regulated genes in the squid.

Additional Thoughts

While this paper is eye opening, it evaluates the squid based on the bacteria. The study’s strength comes from its ability to show similar transcriptions in both the eyes and the light organ. The author’s use of multiple methods to both visualize and quantify gene expression proved very powerful in showing significant transcription differentiation between tissue areas and between differentially colonized squids.  Additional follow up would be to see this from the opposite side – what genes are required by the squid to be colonized by the bacteria. If we removed dac or six with a gene knockout, would the light organ still be colonized?

Citations

1. Peyer S, McFall-Ngai M, Pankey M, Oakley T. Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cuesMechanisms Of Development [serial online]. February 1, 2014;131(1):111-126. Available from: Scopus®, Ipswich, MA. Accessed April 3, 2014.

2. Visick K, Ruby E. Vibrio fischeri and its host: it takes two to tango. Current Opinion In Microbiology [serial online]. December 2006;9(6):632-638. Available from: Academic Search Complete, Ipswich, MA. Accessed April 3, 2014.

3. http://thefeaturedcreature.thefeaturedcreat.netdna-cdn.com/wp-content/uploads/2012/10/Hawaiian_bobtail_squid042.jpg

4. http://microbewiki.kenyon.edu/index.php/File:Vibrio_fischeri_1145457864.jpg

Leave a Reply

Your email address will not be published. Required fields are marked *