Zac Swider - Week Three in the Maslakova Lab

The past week in the Maslakova lab has been full of new discoveries and exciting plans.  By far the most interesting development has been the identification of an obvious phenotype (a physical characteristic) of pilidium larvae lacking a very specific transcription factor.  These larvae were depleted of this protein by injecting a number of zygotes (fertilized eggs) with a morpholino designed to eliminate the production of this protein specifically.

First a little background about what this protein does, and why we care:

A transcription factor binding to DNA

This transcription factor has been associated in a variety of developmental pathways in fruit flies, mice, and humans.  Transcription factors are proteins that bind to DNA, usually with relatively high specificity to a particular region of DNA, and either enhance or repress the expression of proteins coded by that region of DNA.  This process of "what proteins get produced where, and in what quantity" dictates a great deal about the development of a particular organism.  Thus, this protein and similar transcription factors are of great interest to developmental biologists in their quest to tease apart the mysteries of exactly how a single cell can turn into a million cells. Each are ordered in just such a way that an unbelievably complex organism can be formed. The image to the right depicts a transcription factor binding to DNA (image from Wikipedia.com).

Pilidium larva with juvenile worm

As it turns out, this transcription factor is heavily expressed (produced) in the axillary regions of the developing larva.  As I mentioned in a previous blog post, the cells in these axillary regions divide frequently and continuously to produce the bulk of the larval body.  They are also the source of the juvenile rudiments that will eventually become the outside of the juvenile worm.  Remember - the larval body develops first, and then the juvenile worm develops within it before breaking free in a "catastrophic metamorphosis" (see first blog post for video).  Thus, this gene was chosen as a target in attempts to elucidate exactly how these axillary cells pattern both the larval body, as well the developing juvenile body.  The image to the left (courtesy of Svetlana Maslakova) shows a pilidium larva containing a fully developed juvenile worm within it.

However, transcription factors are fairly ubiquitous within the genome of any given organism - so why this specific one?  Simply put - this gene had already been cloned in M. alaskensis by Ph.D. student Laurel Hiebert and this made it very easy to order a morpholino to knock it out.  Furthermore, morpholino knockouts have never been tried before in M. alaskensis, and oftentimes embryos can be extremely resistant to morpholino treatment. This transcription factor seemed as good a place to start as any, considering that we were not 100% sure that the morpholinos would even work.  However, morpholino treatment seems to be working extremely effectively and we may even be able to target other developmental pathways before the end of the summer.

This coming week will be largely spent addressing the phenotype identified in the larvae that we have depleted of this protein.  I will likely be using more specific staining techniques and confocal microscopy to determine exactly what is going on in depleted larvae, compared to normal healthy larvae.  We will also be continuing to address the phenotype identified in MKLP1 depleted larvae.  As I mentioned in a previous blog post, MKLP1 is a motor protein crucial for a cell to identify its mid-line and undergo cell division.  We will also be using fluorescent markers to identify any irregularities in these cells before they lose the ability to undergo cell division - these irregularities will provide clues as to what this protein does and how it interacts with other players in the cell division process.

Time-lapse sequence of cell division - photo by George von Dassow, senior research associate at the OIMB

The image above, taken by George von Dassow (my adviser), illustrates the process by which we will be identifying the affects of MKLP1 knockout.  A similar, though wider scaled, approach will be used to analyze the effects of the transcription factor knockout.  The image above depicts a single cell as it undergoes the process of cell division.  The yellow/orange labeled filaments are microtubules - a dynamic structural component of the cell cytoskeleton that is used during cell division to pull apart the newly duplicated chromosomes.  The chromosomes (DNA) in question are labeled with blue in the above photo.

More results to come soon!