Mock Grant Proposal - An Evo Devo Study of the Cephalopod Chromatophore

Nov 25

The many novel traits of Cephalopods, such as the extensive central nervous system, and the chromatophore structure, have only come under the lens of major research in the past 15-20 years. The unique traits of Cephalopods can potentially provide great insights into how novel traits can develop. In order to further our understanding of these novel traits though, it is always best to start from a system that is already well characterized and studied. The chromatophores (more specifically the melanocytes) in Zebrafish, Danio rerio, show strikingly similar characteristics to those of cephalopods. For this study I will use a candidate gene approach in studying the development of chromatophores in both Cuttlefish, Sepia officinalis, and Octopi, Octopus vulgaris. I will do this by looking for expression of two important Zebrafish chromatophore developmental signals Mitf, and Kit, in the chromatophores of S. officinalis and O. vulgaris. Florescence in-situ Hybridization (FISH) will be utilized in order to map expression of these genes in the cephalopods then, if expression is seen, Crispr-Cas 9 will be used to knockout said genes in each species as a check on the results of the FISH tests.

Background & Significance:

In the phylogeny of Metazoa, Deuterostomia, where Zebrafish are place, and Mollusca, where cephalopods are placed, are consistently within the Nephrozoa branch of Bilateria as can be seen in Figure 1 (Hejnol, 2009). Both Hejnol (2009) et. al. and Philippe (2009) et. al. have shown this relationship to be correct. This signifies a fairly divergent relationship to each other, which can be seen in the many functional differences between the two phyla. As functionally different as they may seem though, a few of the organisms in each phyla have a strikingly similar method of camouflage and patterning - that being Zebrafish (Danio rerio), Cuttlefish (Sepia Officinalis), and Octopi (Octopus Vulgaris). This camouflage comes about through a combination of pigment as well as reflector cells that act to change the color of the animal to its surroundings. Although some of the same cells are present in each of the species, the convergence of the camouflage and patterning systems are more large scale than they are small (Demski, 1992). This is because the direct mechanisms through which they are controlled are almost perfectly opposite to each other.

In Zebrafish, the pigment cells (melanocytes) are singular cells derived from the neural crest and controlled through the endocrine system by different hormones that each cause varied levels of contraction or expansion (Mellgren, 2002; Logan, 2006). The reflector cells (iridophores) are also derived from the neural crest. However, they are controlled directly by neurons instead of secreted hormones. When these cells are subjected to something like norepinephrine, they will dramatically change the coloration of the fish (Oshima, 2002). This system allows the iridophores to react significantly faster than the melanocytes because of the direct control mechanism.

The pigment cells (chromatophores) of Cephalopods on the other hand are much more complex and yet have the same basic function as melanocytes in Zebrafish. Figure 2A represents the structure of the cephalopod chromatophore in the squid Loligo opalescens (Cloney & Florey, 1968). In this you can see the multiple different cells encompassed in this structure including striated muscles, neurons, glial cells, and a sacculus (sack) of pigment (Messenger, 2001). All of these work together as one neuromuscular organ in order to change the color of the cephalopod’s skin. The function of this complex can be seen in Figure 2B showing that as the radial muscles contract the succulus expands, making the chromatophore larger and the pigment visible. When the radial muscles relax however, the sacculus retracts and the chromatophore becomes less visible and thus the animal does not show that pigment (Messenger, 2001). To show even more divergence, this expansion has been shown to be mediated almost solely through neuropeptides with almost none of the classical neurotransmitters affecting them (Loi, 1996). This is obviously very different from Zebrafish since the cephalopod pigment complex is multiple different cell types that are all (directly or indirectly) controlled through the nervous system. The reflector cells in cephalopods (also iridophores) have been shown to be controlled by hormones, specifically acetylcholine, which is acting as a hormone in this instance (Hanlon, 1990). With this it is easy to see that the same basic functions are being carried out in both Zebrafish and Cephalopods however they are happening in completely opposite ways (depending on whether they are neurally or hormonally controlled).

Another functional difference in these systems (that leads to a convergent function) is the pigment that is used. In Zebrafish, the melanocyte cells convert tyrosine into melanin which gives off a dark brown pigment (Camp, 2001). This is in contrast to cephalopods which mostly use ommochromes which are small pigment granules derived from tryptophan (Messenger, 2001). Again, both systems have very different mechanisms, and yet, end up with close to the same result.

In cephalopods, chromatophore cells are not derived from a neural crest like they are in Zebrafish. Although the chromatophores in both species do not have the same origin, the patterning of the cells on a microscopic level are extremely similar. In the cephalopod body plan there are 4 distinct “tegumental fields” or regions. Each of these fields develop from multiple “founder chromatophores” that seem to signal for other chromatophores to develop around it (Packard, 1972). In Figure 3B these founder chromatophores can be seen as the large back dots. The interesting thing about Figure 3 though is that two different organisms’ chromatophore patterning are shown. 3B is the patterning of O. vulgaris where as 3A is the chromatophore patterning of a flounder (Packard, 1972). This patterning, when measured and mapped at the cellular level in cephalopods is “too regular” for the cells not to be signaling with each other (Bassaglia, 2013). At the time of Bassanglia et. al. publishing this paper, no specific gene for the development or patterning of the cephalopod chromatophore organ had been identified.

Another very intriguing aspect about the chromatophore patterning in cephalopods is that when damage is sustained to the dermis of the animal, chromatophores will be regenerated and incorporated seamlessly back into the pattern called for in that region (Yacob, 2011). This would suggest that the cells in this region are able to revert back into a developmental state in order to successfully regenerate this tissue with the correct chromatophore patterning.

Since the chromatophore patterning of cephalopods is so complex and appears to be so similar to patterning in many deuterostomes, looking at the expression of different developmental genes in deuterostomes may give some insights into how the cephalopod patterning is set up. In Zebrafish, there are many factors that cause cells to derive from the neural crest however two of these have been shown in different tests to be active in both cell differentiation as well as chromatophore patterning. Mitf along with orthologs of Kit (specifically Fms) have been shown to be crucial in the development and patterning of the melanophores (Curran, 2010; Mellgren, 2002). More importantly, Mitf has been shown to act as an inhibitor to iridophore development since when Mitf gets down regulated in Zebrafish the iridophore cells become over proliferated (Curran, 2010). Fms on the other hand (the closest known Zebrafish homologue to kit) when knocked out, has been shown to severely disrupt the melanophore stripes of Zebrafish. When the function of FMS is restored though (even towards the end of development) the melanophores show almost complete recovery (Parichy, 2003).

The little bit of research that has been done on the patterning of cephalopod chromatophores has already shown some promising results. The only significant work that has been done so far shows that FMRFamide-related peptides (FaRPs) localize to the chromatophore lobes in the brain as well as the dermal tissue where the chromatophores are found (Aroua, 2011). Since these are not genes though, and no significant genes have been identified to control cephalopod chromatophore development and patterning, a very good place to start is with the well studied system of Zebrafish. Even though Teleost and Mollusca are fairly distantly related, the similarity in function of their chromatophore systems warrant an investigation into the two systems molecular mechanisms of patterning.

Hypothesis: Mitf and/or FMS (Kit) will localize to the chromatophores of Zebrafish but not of Cuttlefish or Octopi. When the expression of these genes are knocked out, part or all of the chromatophore expression in Zebrafish will be disrupted.

Experimental Method:

Specific Aim 1: Determine if expression of Mitf and FMS (Kit) is present in the chromatophores of Danio rerio, Sepia officinalis and Octopus Vulgaris by mapping the expressions of homologs of Mitf, and FMS (Kit) with Florescence in-situ Hybridization (FISH). This will be conducted over the course of multiple developmental stages.

Hypothesis: The Mitf and FMS genes will only show expression in all three of the species being tested, but the genes will only be correlated with the chromatophores in Zebrafish.
Rationale: FISH is an easy way to show if certain genes are being expressed in particular areas of the body making it a perfect experiment for this kind of study. Instead of something like PCR were you are only able to see expression levels, FISH allows for the ability to determine where this expression is localized in the organism.
Methods: Florescent anti-sense RNA probes will be created for the transcripts of the Mitf, and FMS genes. When these probes are introduced, if the gene is present and being transcribed, then they will bind to the transcripts and proceed to fluoresce in these specific cells. These probes will be introduced into the embryos at the time of fixation in order to determine the different localizations of each gene. This process will be carried out multiple times at different developmental stages between organogenesis and hatching of all three species (cephalopod stages as defined in Bassanglia, 2013). Zebrafish, D. rerio, will be acting as a control in these tests since previous studies have already shown these genes to be present (Curran, 2010; Parichy, 2003).
Expected Outcomes: Zebrafish will be expected to show expression of Mitf in premature chromatophore cells during neural crest differentiation and FMS (Kit) in chromatophore cells as they are maturing and becoming patterned. In the two cephalopods, Mitf is unlikely to show expression since it is more involved with neural crest differentiation than patterning however, if it were to be present, it would most likely be seen in the early development of the iridophores. FMS is expected to be seen in the premature chromatophores at, or soon after, organogenesis in both Sepia officinalis and Octopus Vulgaris has started since it most likely aids in the patterning of the chromatophores.
Potential Pitfalls: There is a fairly high risk of false positives in this experiment since the genomes of neither O. vulgaris nor S. officinalis have been fully sequenced. This means the anti-sense RNA that are created for the experiments may end up binding to a closely related gene that is not known to be present, causing a false positive in the experiment. This can only be checked in future experiments once the genomes of the two organisms have been sequenced and homologous sequences of these genes are able to be determined. The other major pitfall is that these gene are not guaranteed to be present in cephalopods so if no expression is shown then new gene candidates will need to be found.

Specific Aim 2: Knock out the genes that showed expression in the FISH experiment by using Crispr-Cas 9 in order to observe any changes in the chromatophore development. Raise the three species to the same developmental stages as was done in the first experiment so a progression of the chromatophores can be seen.

Hypothesis: If expression of either Mitf or FMS in cephalopods is observed in the FISH experiment, then knocking out the gene with Crispr-Cas 9 will yield a disruption in the development of the chromatophores.
Rationale: The results seen with the FISH test allow us to know if the genes in question are present or not. The easiest way to determine if these genes have a functional role in the development of the chromatophores is to destroy their function and observe changes in the development. Crispr-Cas 9 was selected as the method for knocking out any gene expression seen in the FISH experiments. Crispr-Cas 9 is a very precise tool derived from a prokaryotic immune response to modify the genome of an organism (Sanders, 2014). For this reason it was selected in order to modify the genome so there is very little risk of anything else getting modified.
Methods: Zygotes of each species will be raised in accordance with the developmental stages used in the FISH test. In most of the zygotes, Crispr-Cas 9 will be used to knock out the genes that were seen to be expressed in the FISH experiment. Crispr-Cas 9 works by delivering the Cas9 protein and guide RNAs from a prokaryotic immune response into a cell by electroporation in order to splice out a very specific segment of a genome. This splicing will knockout the expression of the gene that was being targeted (Sanders, 2014). Some zygotes will be kept unmodified but will still go through electroporation in order to maintain a control sample in the test. All of the zygotes will then be allowed to proliferate according to the normal developmental plan and will be fixed and collected in accordance with the developmental stages used in the FISH experiment. The chromatophores at each stage will then be examined using fluorescence microscopy to determine any change that may have happened to the chromatophores over the course of development.
Expected Outcomes: If expression of Mitf is seen in the premature or mature chromatophore cells during the FISH experiments then the knockout of Mitf will cause an over-proliferation of the iridophores (Curran, 2010). If FMS is expressed in the premature or mature chromatophore cells however, a knockout will have the expected outcome of sever disruption in patterning of the cephalopod chromatophores. If both genes are found to be expressed then the expected knockout outcome will be a combination of the two previously discussed outcomes (high proliferation of iridophores and disruption of the chromatophores). If expression of Mitf or FMS is found outside of the chromatophore cells then patterning of the chromatophores is expected to not be disrupted when the genes are knocked out with Crispr-Cas9.
Potential Pitfalls: The same pitfalls apply for this experiment as for the FISH experiment. Since the genomes of neither O. vulgaris nor S. officinalis have been fully sequenced, Crispr-Cas 9 has a high likelihood of accidentally disrupting another gene that could greatly affect the results. In order to double check any results seen in this experiment, we would again need to know the full genome of each organism to determine if we were in fact affecting the homologs of the target genes.

References Cited:

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Bassaglia, Y., Buresi, A., Franko, D., Andouche, A., Baratte, S., & Bonnaud, L. (2013). Sepia officinalis: A new biological model for eco-evo-devo studies. Journal of Experimental Marine Biology and Ecology, 447, 4-13.

Bassaglia, Y., Bekel, T., Da Silva, C., Poulain, J., Andouche, A., Navet, S., & Bonnaud, L. (2012). ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca—Cephalopoda). Gene, 498(2), 203-211.

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