Wednesday, August 28, 2013

Zac - summer research recap

A quick recap of the summer’s research: I have been working in the Maslakova lab under my immediate adviser, Dr. George von Dassow, and Ph.D. student Laurel Hiebert. The Maslakova lab is very interested in studying the evolution and development of the pilidium larva, a distinctive larval form of ribbon worms (Phylum Nemertea) in the clade Pilidiophora. This larval form originally develops as a highly ciliated, hat-shaped larva, which eventually develops into a juvenile worm within its blastocoel. This juvenile worm forms as a result of the growth and intercalation of a number of distinct stem cell bundles (imaginal disks) that eventually fuse around the larval stomach. The result of this developmental pathway is a swimming larval form containing a digestive system that terminates inside of the juvenile worm. Since the larva and juvenile are essentially connected at the mouth, the transition from free-living larva to free-living juvenile culminates in a "catastrophic metamorphosis" in which the juvenile inverts the larval body and swallows it in the process. This mode of development is unique in the animal kingdom, and the fact that it appears to have evolved from a direct developing ancestor make it all the more interesting.

Pilidium developmental sequence, age increases to the right - images by Svetlana Maslakova
In order to study the evolution and development of the pilidium larva more thoroughly, the Maslakova Lab has identified Micrura alaskensis as a model organism. M. alaskensis makes a good model organism because it is readily available and easy to keep alive in the lab. The oocytes are easily obtained and fertilized externally and the resulting larvae can be reared through metamorphosis in a reasonable amount of time (six to eight weeks). Dr. Svetalana Maslakova has already done a very thorough job describing the development of M. alaskensis, and Ph.D. student Laurel Hiebert has recently constructed a high quality developmental transcriptome, which are essentially a list of genes that are expressed in the early development of this species. My summer project involved inhibiting the translation of four of these genes by using morpholino injections. Morpholinos are a fairly common tool in modern molecular biology to study genes by using a "loss of function" approach. This approach is somewhat analogous to studying an automobile assembly line by kidnapping a worker at the beginning of each work day and seeing what happens to the cars at the end of the day as a result. If the cars came out of the factory at the end of the day missing the rear windows then one could reasonably argue that the worker in question was responsible for installing these windows; and furthermore, that the installation of the rear windows is distinct from that of the front windows (logic by Bill Sullivan).

Morpholino treatments have never before been performed in this species of nemertean, and so one of the first big questions was simply: "Will morpholino treatments work?" We proceeded to tackle this question by performing a positive control: an experiment with a known outcome used to determine if things are working as we had hypothesized. In order to do so, we repeated an experiment published by Henry & Martindale (2008) that involved knocking down the production of beta-Catenin in a related nemertean - Cerebratulus lacteus. Henry and Martindale reported a lack of gastrulation and excessive apical tuft formation in C. lacteus. We were easily able to distinguish these same features in M. alaskensis, validating the assumption that the morpholinos we are using are targeting the genes that we think they are targeting.

b-Catenin morphant phenotype in C. lacteus (left) vs b-Catenin morphant phenotype in M. alaskensis (right)
Our next target was the Intra-Flagellar Transport 88 (IFT88) gene. The IFT88 gene product is supposedly crucial for the formation of cilia, and since M. alaskensis larvae are covered in cilia, we thought that it would be awfully nice to be able to knock out cilia production for the purposes of live (video) imaging if nothing else. Unfortunately, this morpholino did not work in the way that we expected it to work. Not only did we observe more or less perfect cilia formation but the larvae ultimately developed a nonspecific and overall unhealthy looking phenotype. The most likely explanation for this is that the morpholino knockdown was not 100% effective—we know for a fact that it never is—and that these larvae are still capable of developing cilia with whatever remaining IFT88 protein that manages to be produced. It is important to note that this result does not actually contradict our positive control. This experiment has never been performed previously and it is impossible to say that the nonspecific—and generally unhealthy—phenotype resulting from this morpholino treatment was due to nonspecific morpholino toxicity and not IFT88 knockdown. Since this morpholino did not do what we thought it would do, we decided to abandon it for the time being and move onto bigger and better things.

The next gene of interest is called the Mitotic Kinesin-Like Protein 1 (MKLP1) gene. The MKLP1 protein is crucial for cell division in all animals previously studied. That being said, clearly there must be some amount of it provided in the oocyte prior to fertilization in order for first cleavage to occur. Since we can only knock down the production of future MKLP1, we were curious to see how many cleavages our morpholino treated embryos could undergo before they depleted the endogenous MKLP1 and failed to complete cytokinesis. The movie below shows a 16-32 cell M. alaskensis embryo attempting to undergo cell division and failing. If you watch the bottom cells closely, you can see the cells almost complete cytokinesis before they fail to achieve abscission and relapse to become one cell instead of two. This is characteristic of MKLP1 depletion as seen in human cells, and other animal cells, and indicates that the endogenous MKLP1 present in these oocytes is only sufficient for three or four cleavages before it is depleted.

The brightly lit cells near the top of the frame serve as a positive control. These cells never received morpholino treatment and subsequently have no problem undergoing cell division.

video

I have also spent a great deal of time learning to use the confocal microscope to image both fixed and live larvae stained with various fluorescent markers. By using fluorescent stains that not only bind to different tissues or sub-cellular components, but also fluoresce at different wavelengths of light, we can use the confocal microscope to put together high quality images with different tissues or sub-cellular components marked with different colors.

M. alaskensis Pilidium Larva - Oral View (Image by Zac Swider)
For example, the image above shows a confocal z-projection of a M. alaskensis pilidium larva (oral view) composed of about 130 individual images stacked together. This compilation of images shows actin filaments (stained with phalloidin) highlighted in grey, DNA (stained with Hoechst 33342) highlighted in light blue, and cells actively undergoing division (stained with an anti-phospho-histone antibody) highlighted in bright green. I have been using these staining techniques, in concert with the confocal microscope, extensively over the past couple weeks in order to more thoroughly characterize the effects of one particular morpholino treatment. Unfortunately, due to the sensitivities of unpublished data, I will not be going into any more detail than that. Come to the annual SICB conference in Austin, TX, Jan 3-7 2014 to hear the whole story!

1 comment:

  1. Here are two techniques that might produce a stronger knockdown of IFT88, one inexpensive and the other involving synthesizing another Morpholino. First, try autoclaving the Morpholino stock prior to setting up the injection, or at least heating the stock to 80C or so. If there are Morpholino oligo complexes that have formed in the stock and decreased the oligo efficacy, heat can usually disrupt the complexes and release single-stranded oligo. If you are going to autoclave, put some colored water (the same volume as your MO stock) in the same kind of container as used for your MO stocks and run the colored water through the autoclave to test whether your autoclave causes volume changes. Removing the container and cooling it right after return to 1atm pressure might help prevent volume changes. The second technique is to get a second non-overlapping oligo targeting IFT88. This can be used as a specificity control, comparing its elicited phenotype with that elicited by the first oligo. Co-injection of the two oligos should produce dose synergy and give a much stronger knockdown.

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