Syncytia are multinucleated cells that form distinct functional compartments. In a new paper in Development, Charles Ettensohn and colleagues use the sea urchin embryonic skeleton to study how specialized compartments are generated in a syncytium to give rise to local skeletal patterns. We caught up with first author Jian Ming Khor and corresponding author Charles Ettensohn, Professor at Carnegie Mellon University, to find out more about their research.
Jian Ming Khor (left) and Charles Ettensohn (right)
Charles, can you give us your scientific background and the questions your lab is trying to answer?
CE: I'm a developmental biologist by training and at heart. Much of our work over the past 10 years has been aimed at understanding gene regulatory networks that establish cell identities during embryogenesis and linking those networks to morphogenetic processes, using sea urchins and other echinoderms as experimental models.
Jian Ming, what brought you to join Charles' lab and what drives your research today?
JK: I was drawn to Chuck's group because of the research focus of the lab, which is to understand how the process of embryonic development is encoded by the genome. During my lab rotation, I was also very much captivated by the model organism the lab uses, which is the sea urchin. I was also interested in using sea urchins to study evolutionary developmental biology. The sea urchin belongs to the echinoderm phylum, which consists of five classes of organisms with diverse developmental programs and body forms, making the group perfectly suited for evo-devo studies. Finally, and probably most importantly, I was drawn to Chuck's mentorship style. He gave me a lot of freedom in pursuing research questions that I am interested in and was always available to give advice and guidance whenever I needed it. Currently, I am a postdoctoral fellow at the National Institutes of Health in Dr Brant Weinstein's lab, where I am working on elucidating the epigenetic regulation of zebrafish fin regeneration.
What was known about the molecular compartmentalization in a syncytium before your work?
CE: There are many examples of syncytia in nature and numerous studies have documented molecular and functional sub-domains within these syncytia. How those compartments are generated is less well understood. In our case, we knew that the skeletogenic syncytium of sea urchin embryos is composed of distinct sub-domains of gene expression based on the heterogeneous distributions of many mRNAs, but we didn't know how those domains were produced or whether they could produce the complex patterns of biomineral growth that we see as sea urchins build their larval skeletons.
Can you summarize the key findings in a paragraph?
CE: We found that transcription factors don't translocate very far from the site of translation within the syncytium. This is due to several factors, including nuclear import sequences and the DNA-binding motifs that these transcription factors contain, which restrict the proteins to nearby nuclei. The limited mobility of transcription factors is very important as it allows distinct gene regulatory states to be maintained within the syncytium. The transcription factors we tested were known from previous work to regulate many genes encoding proteins that regulate the growth and properties of the calcite-based skeleton. That led us to test several of these biomineralization proteins, and we found that these also have very limited mobility within the syncytium. As the skeleton forms, most biomineralization proteins are secreted into a membrane-bound compartment where the biomineral is deposited. We found that the N-terminal signal sequences that target these proteins to the secretory pathway play an important role in restricting their mobility. Based on these findings, we can begin to understand how a syncytium can produce the beautiful, local patterns of skeletal growth that we see during sea urchin embryogenesis.
In your article, you used an optimized Tet-On system your lab developed to conditionally induce gene expression in sea urchins. Can you briefly explain this system and its applications?
CE: The Tet-On system was first developed more than 20 years ago and has been used successfully in several model systems. It has two components: a transcription factor that is activated by binding to tetracycline (or doxycycline) and specific sites in DNA that are bound by the active transcription factor. If a cell or organism contains a gene of interest under the transcriptional control of these DNA-binding sites, and the transcription factor is also expressed, then the gene of interest will only be transcribed when tetracycline is added. This provides exquisite temporal control over the expression of the gene of interest. If the transcription factor (or the gene of interest) is also controlled by a tissue-specific promoter, then the system can also provide spatial control over the expression of the gene of interest. It's a very powerful and versatile method for controlling gene expression.
What are the implications of your findings for the broader understanding of functional compartmentalization in the syncytia of other organisms?
CE: Our findings show that basic mechanisms that control the subcellular trafficking of proteins, such as the localization of proteins to the nucleus by import sequences and DNA-binding motifs, or to the secretory pathway by signal sequences, can produce distinct transcriptional regulatory states and functional sub-domains within syncytia. Because other syncytia also exhibit molecular and/or functional compartmentalization, these mechanisms are likely operating there as well.
Jian Ming, did you have any particular result or eureka moment that has stuck with you?
JK: For the present study, my eureka moment occurred the first time I was able to see GFP expression in sea urchin skeletogenic cells using the Tet-On system. At the beginning, it was a long shot as I was unsure whether doxycycline would be toxic or the transactivator would be inactive in sea urchin embryos. Following that finding, I was ecstatic, as it presented a new and exciting opportunity to conditionally express essentially any gene in any cell we want in sea urchins, as long as a cell-type specific enhancer is available.
I was ecstatic, as it presented a new and exciting opportunity to conditionally express essentially any gene in any cell we want
And the flipside: were there any moments of frustration or despair?
JK: There were plenty of moments of frustration, mainly due to the seasonal reproductive cycle of different sea urchin species. There were times when eggs would have poor fertilization rates and microinjections would not yield any viable embryos.
Where will this story take the Ettensohn lab?
CE: We plan to focus on upstream developmental events. We know that local signals from overlying ectoderm cells produce the compartments of gene expression and skeletal growth we see in the skeletogenic syncytium. One of these signals is vascular-endothelial growth factor (VEGF), but there are other, as yet unidentified, signals. We want to identify the remaining signals and elucidate the molecular pathways by which these local signals impinge on the gene regulatory network that operates in the skeletogenic cells.
Why did you choose to submit your paper to Development?
CE: We've published many papers in Development over the years, as we regard it as a flagship journal in the field.
Finally, let's move outside the lab – what do you like to do in your spare time?
CE: I enjoy hiking and cycling, and I play in a local Cajun band.
JK: My partner and I are movie buffs, and we have an affinity for horror movies. I also enjoy taking long walks with my dog and gaming.
J.K.: National Institutes of Health, Bethesda, MD 20892, USA.
C.E.: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15218, USA.