Graduate school can be a grind. Especially, it seems when you’re in it. It culminates in the defense of a body of work. It is a symbolic event for some students and in some departments. In others it is a true battle. Regardless, the event is ripe with significance. As a student you develop as a learner, you accept the wisdom of your mentor and those around you who are more developed and experienced than yourself. As you continue you become more confident, more knowledgeable, more capable of explaining your opinions, your results, your conclusions. When you are ready to finish, you can challenge your peers who likely include your mentor and other professors. There is an ironic part to this process. Even though it’s stressful and challenges your self-esteem and determination, once it’s said and done it seems like such a simple, pleasant time.

Craniofacial Biology graduate students, Michael Mogass and Larry Zhou both completed this rigorous process and received their Ph.D. diplomas this summer. What follows are descriptions of the work they completed during thier time at the CCMB.

Michael Mogass- desmosomes, the anchor and TGF-b

Though Michael Mogass came far to get to Southern California from Ethiopia he didn’t come here directly. As a student awaiting an undergraduate education, Mogass received a scholarship opportunity to study abroad in Russia. At the time, relations between Ethiopia and Russia were better than that between Ethiopia and the U.S. But as the relations between his home country and the U.S. improved, Mogass took the opportunity to come to Southern California where he completed a master’s degree at Cal State Los Angeles. He then came to study under the tutelage of Dr. Charles Shuler.

Shuler’s research group focuses on the genetic control of palate formation. This developmental process, which goes awry in as many as 1:600 live births, results in a disfiguring cleft of the palate and/or lip. As one of Shuler’s students, Mogass took ownership of a project that centered on the role of the desmosomal adhesion complex in palate formation.

An intact palate is generated when two embryonic tissue projections in the developing mouth come together, fuse and form one confluent tissue process. In order for the palate to fuse, the two palatal tissue processes must adhere, in part, to resist the pulling force that is generated by the lateral growth of the rest of the embryonic head.

The tips of the palatal processes are not held together with some glue-like substance but with molecular anchors of sorts. Desmosomal complexes are composed of several proteins (i.e. desmocollin, desmoglein, desmoplakin and plakoglobin) that assemble into a plaque and span the membrane of the cell. Cells that come together and adhere, as the cells on the tips of the palatal shelves do, are adjoined via these membrane spanning desmosomal plaques.

Mogass characterized the molecular components of desmosomes in the fusing palate. He established the timing, localization and overall make-up of desmosomal proteins in the palate and compared these patterns to those found in other tissues that adhere during development. The onset of desmosome production, in and of itself, is critical says Mogass. If there’s no adhesion there’s no fusion. If there’s no fusion it leads to a cleft palate, he explains. In other systems, he continues to describe, certain components of the complex, like the membrane spanning proteins, desmoglein-1 and desmocollin-1, have variants that are produced simultaneously. In the palate however, these co-expressed variants are not found.

Mogass next determined whether or not desmosome composition was affected in TGF-b3 knock-out mice. Removal of the powerful growth factor, TGF-b3, from the mouse genome had previously been found to dramatically increase the incidence of cleft palate. It was also known, as Mogass explains, that the addition of TGF-b3 was associated with an acceleration of palatal fusion. Mogass analyzed the TGF-b3 mutant mice and noted that the expression of essential demosomal components was substantially reduced. When pre-fusion palatal shelves were removed from the TGB-b3 transgenic mice, put in vitro, and treated with TGF-b3, Mogass observed that the fusion process seemed to speed back up to a normal rate. As he measured the amount of desmosome production, he found that the levels had also been replenished. Though the experiments Mogass performed implicate a role for TGF-b in regulating desmosome production, he posits a number of possibilities for how this could occur. We don’t know which component of the complex is specifically affected, one or all, or whether it affects the transport of one of these factors, plakoglobin, from the nucleus to the cell membrane, says Mogass.

Mogass is currently continuing his work with Shuler trying to find new ways to overcome some of the drawbacks of performing palate studies. One such factor that hampers palate studies is the fact that the overlying epithelial cells covering the palatal shelves are few in number. Changes in gene expression are difficult to measure because of the small signal they produce relative to surrounding cell populations. To this end, Mogass and Shuler have initiated a collaboration with a nearby hospital in order to use their laser capture micro-dissection apparatus. This tool will enable the two to isolate single epithelial cells from the palate, measure their patterns of gene expression and perhaps even grow them in culture to study their behavior.

Mogass M, Bringas P Jr, Shuler CF. Characterization of desmosomal component expression during palatogenesis. Int J Dev Biol. 2000 Apr; 44(3): 317-22.

Larry Zhou- the ins and outs of amelogenin production

Larry Zhou hit the ground running with his CBY Ph.D. Having received his undergraduate degree from Fu Dong University in China, Zhou next went to work as a research assistant at the Shanghai Institute of Cell Biology, equivalent to our own NIH. He came to the U.S. to start his doctoral studies, spent two years at Indiana University and soon found himself drawn west to USC. His decision was no doubt influenced by the fact that his wife, Xin-Wen, was already a graduate student studying at USC.

Zhou looked for an interesting project and found a match for himself with CCMB’s Malcolm Snead. Since I had a relatively strong molecular biology background, he encouraged me to take a project doing promoter bashing on the amelogenin gene. When I joined the lab I had a piece of the amelogenin promoter. The group had just made a transgenic mouse with that promoter fragment and found this region was sufficient to direct amelogenin expression. My job was to narrow down the promoter to find some critical region and transcription factor that would bind to that region and would be responsible for regulating amelogenin expression, said Zhou.

Zhou spent a short three years completing his Ph.D., a timeframe made possible by his hard work and strong background. Despite this, his first eight months were frustrating, It felt like the project was going nowhere, he admitted. Zhou searched for critical promoter regions by looking at clones that had been picked up with a screen using a 500 DNA base-pair piece of the amelogenin promoter. At the time we didn’t know where to go, said Zhou. But the group also generated deletion constructs of the promoter, put the constructs into an ameloblast cell line and found they could direct the amelogenin expression with particular constructs. We did a computer search to see what known transcription factors could bind the amelogenin promoter sequence in this region and found C/EBPa, explained Zhou.

Zhou reintroduced C/EBPa in excess amounts to ameloblast cells and found the transcription factor increased expression of amelogenin. He also discovered that the positive effects of C/EBPa on amelogenin expression were counteracted by another transcription factor called Msx-2. In isolation, Msx-2 appears to repress amelogenin expression. Interestingly, Zhou found these two transcription factors worked to antagonize one another in ameloblasts. I tried to understand the mechanism behind this relationship, said Zhou. What he found was that the two factors weren’t competing for the same site on the amelogenin promoter but rather that C/EPBa and Msx-2 interacted with each other at the protein level. Thus, if one factor was present and the other was not or if one were in more abundance than the other their effect over transcription would dominate, be it positive or negative. 

Though the above work provided closure for his dissertation project, Zhou continues his work on the project as a post-doc. Zhou hopes to further unravel C/EPBa’s role in mediating amelogenin expression. Now, we’re testing the C/EBPa knock-out mice to see if they have a phenotype in ameloblast or enamel formation. We’re also looking to see what signaling pathway is responsible for upregulating C/EBPa expression in ameloblasts, said Zhou.

Zhou must balance a number of weighty issues when making a decision about his future as a biologist. Moving to a new laboratory is a tactical risk. Everything has to be right, says Zhou, the relationship with a new advisor, a new project. I need to keep my options open, said Zhou. Part of this entails pursuing his interests in a burgeoning biotech field called bioinformatics. As Zhou explains bioinformatics uses computer and mathematical methodologies to study issues in biology. Right now Zhou is enrolled part-time in USC’s new Bioinformatics master’s program. Zhou intends to direct his academic efforts to the computer science portion of the program because he explains, I have a strong background in molecular biology, but says Zhou, I think I’m a little too old to learn mathematics and design algorithms for biology.

Zhou YL, Lei Y, Snead ML. Functional antagonism between msx2 and CCAAT/Enhancer-binding protein alpha in regulating the mouse amelogenin gene expression is mediated by protein-protein interaction. J Biol Chem. 2000 Sep 15; 275(37): 29066-75.

Zhou YL, Snead ML. Identification of CCAAT/enhancer-binding protein alpha as a transactivator of the mouse amelogenin gene. J Biol Chem. 2000 Apr 21; 275(16): 12273-80.