Eye Specification Genes in the Bacterial Light Organ of the Bobtail Squid

Figure 1: Euprymna scolopes, the Hawaiian bobtail squid

Figure 1: Euprymna scolopes, the Hawaiian bobtail squid

WHY USE EUPRYMNA SCOLOPES AS A MODEL ORGANISM?

Euprymna scolopes, the Hawaiian bobtail squid, serves as an important model organism due to the beneficial symbiotic relationship it shares with Vibrio fischeri, a bioluminescent bacterium. The relationship aids in understanding interactions between bacterial cells and a host, as well as the immune system of the host’s ability to specify between helpful and harmful bacteria. The selectivity and specificity of both partners is also of note, as V. fischeri seek out the squid for colonization, while the squid forms a one-on-one symbiosis with the bacteria. Rather than residing in the intestinal tract of the host, however, V. fischeri colonizes a squid’s light organ, a unique location and an important factor in this discussion.

INTRODUCTION:

The bobtail squid is an organism with both a complex set of eyes, as well as a photophore, or light organ. The bioluminescent effect of V. fischeri in the squid’s light organ acts to camouflage the squid by mimicking moon and starlight, a tactic known as counter-illumination. This aids the squid in remaining unseen by predator and prey alike. The bacteria are harvested via pores lining the light organ, which in many aspects strongly resembles the organism’s eyes. Both include “a lens with crystallin proteins, genes and proteins involved in phototransduction and the physiological ability to respond to light (Peyer).” The ink sac, which surrounds the photophore, functions as an iris and choroid and could potentially aid the light organ in detecting ambient light, thus controlling the amount of light emitted by V. fischeri.

 While the eyes and light organ develop from different germ cell layers (ectoderm and mesoderm, respectively) and at different stages, light stimulus is important for the development of both tissues. While the eyes rely on environmental light for maturation, the light organ is influenced by endogenous light emitted from V. fischeri, among other bacterial cues. The host-symbiont relationship allows for the examination of the effects on ocular-like development tissues within the light organ, via bacterial cues.

PURPOSE OF STUDY:

In the main paper cited for this topic, researchers examined whether certain eye specification genes, pax6 (paired box gene 6), eya (eye absent), six (sine oculis) and dac (daschund) were expressed in the light organ during embryogenesis, as well as any symbiotic regulation during postembryonic development.

Figure 2: (A) Regulatory network of eye-specification genes, (B) Juvenile E.scolopes with light organ identified, (Peyer, et. al.)

Figure 2: (A) Regulatory network of eye-specification genes, (B) Juvenile E.scolopes with light organ identified, (Peyer, et. al.)

METHODS:

The embryos and juveniles used in this study were produced from wild-caught squid. While previous studies had already shown the genes in question had in fact been expressed in the light organ tissues, researchers sought to amplify cDNA from both eye and light organ tissues using standard PCR to determine if the same genes were expressed in both tissues, along with protein sequence analysis to determine if functional domains were conserved.

Using in situ hybridization (ISH), expression patterns of the four gene transcripts were examined during embryogenesis (eyes) and early postembryonic development (light organ). Samples were taken from a single clutch of eggs to minimize variation and eye development was observed at four stages (~1/4, 1/2, 3/4 and nearly hatched). Reverse transcription PCR was used to confirm the presence of each transcript at every desired stage. To study the effect of V. fischeri on transcript expression in the postembryonic light organ, four sets of juvenile squids were used: newly hatched wild-type (WT), uncolonized (UCO), colonized (CO) and those colonized but with a V. fischeri mutant defected for light production (lux). To determine the effect of bacterial light on gene expression, WT samples were compared to lux samples. To determine if other bacterial cues affected gene expression, WT and lux samples were compared to UCO samples.

RESULTS:

Evidence was obtained indicating expression of full-length transcripts for all target genes in both the eyes and light organ. Because the eyes develop earlier than the light organ, gene transcripts were localized in the eye primordia and across the skin at ~1/4 embryogenesis (Stage 18). At ~1/2 embryogenesis (Stage 22), the transcripts had localized to the eye, optic lobe and tentacles and fins (excluding eya). At ~3/4 embryogenesis (Stage 26), pax6 and six transcripts were present in the optic nerve and olfactory organ (along with dac). Also at this time, all transcripts were present in various other regions including the optic lobe.

Figure 3: Target gene expression at various stages with regions labeled (e:eye, ol:optic lobe, t:tentacles, s:statocysts, sg:shell gland, m:mantle, f:funnel fold, g:gill, f:fin, n:nuchal organ, on:optic nerve and o:olfactory organ), (Peyer, et. al.)

Figure 3: Target gene expression at various stages with regions labeled (e:eye, ol:optic lobe, t:tentacles, s:statocysts, sg:shell gland, m:mantle, f:funnel fold, g:gill, f:fin, n:nuchal organ, on:optic nerve and o:olfactory organ), (Peyer, et. al.)

The gene transcripts were also localized and discernable in the light organ via ISH at ~Stage 26 (excluding eya) and by Stage 29, all four transcripts had localized in the anterior appendages and remained visible in the pores (again, excluding eya). After hatching, all transcripts were visible in the anterior and posterior appendages, around the pores, and excluding eya, in the ciliated ridges as well.

Figure 4: Target gene expression in the light organ at various stages with regions labeled (hg: hind gut, is: ink sac) and arrows indicating transcript signaling localization (grey: anterior appendages, brown: posterior appendages, blue: ciliated ridges and green: pores), (Peyer, et. al.)

Figure 4: Target gene expression in the light organ at various stages with regions labeled (hg: hind gut, is: ink sac) and arrows indicating transcript signaling localization (grey: anterior appendages, brown: posterior appendages, blue: ciliated ridges and green: pores), (Peyer, et. al.)

 

 

 

 

 

 

 

 

 

 

Upon colonization with V. fischeri, the genes showed a loss of expression in the light organtissue as well as varying responses to the bacteria. pax6 showed loss of signaling in the WT, CO and lux sample light organs relative to the UCO. No significant differences were observed in pax6 expression in the WT, CO and lux samples, indicating a non-light induced bacterial effect on its expression. The same results applied to eya expression. Significant loss of signaling was observed for six in WT and CO samples, but not lux relative to UCO, with little differing between lux and UCO. These results indicate a light induced bacterial effect on six expression. Relatively insignificant loss of signaling was observed in all samples regarding dac expression.

Figure 5: Target gene expression in each of the four samples (WT: wild type hatchling uncolonized, UCO: uncolonized, CO: colonized and lux: lux mutant), (Peyer, et. al.)

Figure 5: Target gene expression in each of the four samples (WT: wild type hatchling uncolonized, UCO: uncolonized, CO: colonized and lux: lux mutant), (Peyer, et. al.)

ISH was also performed on sectioned samples to determine if the target genes were expressed in light organ crypts that directly interacted with V. fischeri. All transcripts were observed at 24 hours after hatching and were not noticeably altered by symbiosis. Their presence an additional 48 hours later suggests a role in phototransduction development within the first few days after hatching.

DISCUSSION:

Based on the study, all eye specification genes were expressed in both the eye and light organ of E. scolopes. Within the light organ, however, gene expression depended on varying bacterial cues. These findings suggest that the target genes, among others, are subsequently regulated by the bacteria and have an impact on light organ development. In previous studies, while the four genes had been identified in other mollusks, none had been isolated in a photophore, and eya, six, and dac had not been described within the genome of a cephalopod.

The study also noted expression of the target gene transcripts in non-optical tissues such as the olfactory organ, statocysts (balance sensory receptor) and the skin of E. scolopes. The transcripts may also play a role in the development of chromatophores (light interacting structures). The study also illustrated the presence of target genes in the light organ of E. scolopes, one of only a few organisms that use bacterial photophores. In essence, the study was the first to show the eye specification genes presence in photophores. Mutual expression of the genes between the eyes and light organ suggest the importance of said genes “in the development of light interacting tissues regardless of embryonic origin (Peyer, et. al.).”

Along with the eye specification genes, many phototransduction genes and proteins were shared between the eyes and light organ. In the eyes, these proteins respond to ambient light, while in the light organ, they respond to bacterial induced light. The expression of eye specification genes could potentially drive the expression of phototransduction genes in the light organ, leading to physiological responses. Also noted in the study results was the role pax6 and six play in eye lens development, and the possibility that these genes may also be used to form the light organ lens.

CONCLUSION AND USES IN FUTURE STUDIES:

Based on the data obtained, all four target gene transcripts essential for eye development were found to be expressed in the light organ of E. scolopes. The similarity between the two structures provides evidence of the expression of the target genes in “two non-homologous tissues that differ in organismal function (Peyer, et. al.).” The results also propose further questions regarding the bacterial and host immune system interaction and their effect on development of the light interacting structures.

 The results of the study illustrate several ways that multiple organs can respond to and interact with light. Future study of the coordination of eye specification genes and phototransduction genes in both the eye and light organ is a subject now more ideally understood. In broader terms regarding the interaction of E. scolopes and V. fischeri, new camouflage techniques can potentially be developed for the military, and a path to understanding the interactions between animal and bacterial cells can be explored, ultimately shining light on potential ways to fight and avoid bacterial infections in humans.

PAPER CRITIQUE:

While the paper presents some interesting findings in terms of the similarities and transcript expression of the target genes in both organs, there is little mention of how the eye specification genes are actually affected by bacterial cues. While the paper goes into length about the discovery of the presence of the gene transcripts in both the eyes and light organ, there is only brief mention of their relationship to the affects of V. fischeri, namely the various genes being affected by either light induced or non-light induced cues. The paper asserts a pseudo-cause and effect relationship between eye specification genes and phototransduction genes but claims further studies must be performed. Lastly, there is little mention of future application; in fact, most of the information presented in the section above came from the embed videos (although the first video involves one of the lead researchers for this paper).

REFERENCES:

Kostic, A.D., Howitt, M.R., Garrett, W.S. (2013). Exploring host-microbiota interactions in animal models and humans. Genes. Dev. 27. 701-718.

Peyer, S.M., Pankey, M.S., Oakley, T.H., McFall-Ngai, M.J. (2014). Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues. Mechanisms of Development. 131. 111-126.

Rader, B.A., Nyholm, S.V. (2012). Host/microbe interactions revealed through “omics” in the symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri. Biol. Bull. 223. 103-111.

*Initial Image obtained from “The Feature Creature” http://www.thefeaturedcreature.com/2010/11/hawaiian-bobtail-squid-and-its-crazy.html

*Figures 2-5 were obtained from the paper by Peyer, et. al.

 

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