Sarah Bashiruddin Georgina Lopez Jillian Merica Sarah Wardlaw

Sarah Bashiruddin Georgina Lopez Jillian Merica Sarah Wardlaw Introduction: Dr. Carol Erickson and her lab study the cellular and molecular mechanisms by which neural crest cells differentiate and migrate to their target locations in chick. The review article, Lineage Specification in Neural Crest Cell Pathfinding (2007) details the two principal models for neural crest cell pathfinding with respect to their differentiation into progenitor cell types, such as melanoblasts, and their resultant migratory patterns. The article Directing pathfinding along the dorsolateral path the role of EDNRB2 and EphB2 in overcoming inhibition looks at melanoblast migratory patterns in further detail. This study investigates the role of the EDNRB2 and EphB2 receptors in permitting melanoblast invasion of the dorsolateral migratory pathway. Our class was interested in the regulation of these receptors as well as other environmental cues involved in cell communication and pathfinding. We had the opportunity to ask Dr. Erickson and postdoctoral student Dr. Melissa Harris about these topics. 1. Michael J.F. Barresi Briefly tell us a bit about yourselves, your career paths over the years, and specifically what led you to begin working on neural crest migration in the chick. Dr. Melissa Harris received her undergraduate degree in Genetics from the University of California at Davis. She worked, on-campus, at a veterinary genetics lab for two years before pursuing a Ph.D in Cell & Developmental Biology studying neural crest cells, specifically melanoblast migration in Dr. Erickson s lab. Dr. Harris is slated to pursue a postdoctoral research at the National Institutes of Health studying genetics and developmental biology. Dr. Carol Erickson received her undergraduate degree in Biology at Wilson College. She then pursued a Ph.D at Yale, where she studied cell motility in culture with her mentor, Dr. John Trinkaus. She did postdoctoral research at the University of Oregon studying the neural crest in mutant mice and pursued the study of neural crest cells in chicks as a newly-appointed assistant professor at UC Davis. Over the course of thirty years, Dr. Harris has observed tremendous scientific progress regarding the molecular models describing neural crest morphogenesis. In response to questions of her experience as a female in science, Dr. Erickson remarked that she has not felt disadvantaged as a woman in her field. She has enjoyed the respect of male colleagues throughout her career, and notes that the balance between her professional and family life were only possible with the support of her spouse and the sacrifice of personal time. 2. Helene Parker I understand that pre-specified melanoblasts have specific receptors which are

upregulated in your model, but by what mechanism does EDNRB2 begin to be upregulated to trigger the advance of melanoblasts from the MSA to the DL pathway? Dr. Erickson identifies the possible roles of transcription factors MITF and FoxD3 in upregulating EDNRB2 expression. These transcription factors regulate melanogenesis by turning on genes responsible for the morphogenesis and production of pigment in neural crest cells. Her lab intends to investigate these transcription factors further by monitoring the onset of expression, the level of EDNRB2 expression with upregulation of MITF and FoxD3, and by identifying which genes are up- or down-regulated with MITF, FoxD3 expression via microarray analysis. 3. Carolyn Cunha I understand that both EBNRB2 and EphB2 receptors are necessary for dorsolateral migration of melanoblasts and that EBNRB2 can rescue the loss of EphB2. Why are both of the receptors necessary? Are both needed in equal amounts or is one upregulated/downregulated more than the other at different times? This research most importantly demonstrates that development is sloppy and will be better understood as more research is conducted. It is not that precise levels of gene (or receptor) expression are needed, but rather that combinations of genes (or receptors) can push certain cells towards migration inhibition or stimulation. These combinations ensure that development proceeds independent of just one protein failing. In melanoblasts, the combination of both EphB2 receptors and EDNRB2 seem to reach a threshold that allows these cells to enter the dorsolateral pathway. If time and resources permit, one way to address this question is to take the expression constructs used in this study and place them under the control of differential strength promoters. For instance, take EDNRB2 under a weak promoter and EphB2 under strong promoter, then assess whether the combination is sufficient to permit cells to invade dorsolateral pathway or vice versa. 4. Sarah Wardlaw Are EphB2 and EDNRB2 responsive to the same inhibitors? Is it possible that EphB2 overexpression inconsistently compensates for EDNRB2 knockdown due to a differential interaction with inhibitors? 5. Sean Burton You have shown that certain levels of EphB2 and EDNRB2 are required for melanoblasts to follow the DL pathway, and that over expression of EDNRB2, and sometimes Eph2, can rescue this ability in shrna knockdown cells. Do these findings eliminate the possibility of another receptor working alongside these ones and why? These questions were combined to answer: why EphB2 does not completely rescue the loss of EDNRB2? First, it could be an experimental artifact. It is possible that the expression construct used for EphB2 is not strong enough and the rescue does not actually reflect the threshold model proposed in her paper. Second, melanoblasts express both EphB2 and other Ephrin receptors. Some other Eph receptors, such as EphB3, respond repulsively to Ephrins present in the doroslateral pathway. Although the

expression of EphB3 is very low in melanoblasts, this low level of repulsion may be just enough to prevent EphB2 from compensating for the loss of EDNRB2. Moreover, there are other receptors involved in pathway choice, such as Robo and Slit. In terms of dorsolateral invasion, melanoblasts downregulate their Robo receptors so that they are no longer responsive to Slits in the pathway. It is important to note that all of these (EDNRB2, endothelins, EphB2, Ephrins, Slits, Robos, and barrier molecules) are present in the pathway, and combine as either positive and/or negative cues to induce dorsolateral migration. 6. Jillian Merica I understand that EphB2 is the specific receptor that mediates the attachment to fibronectin, but you mention that there may be more signaling systems involved in the transition between migratory pathways, like chemoattractants. Could you talk more about these other possible signaling systems, specifically the role ephrin Bs and ET3 play in your proposed model for melanoblast DL migration? Dr. Erickson focuses on the internal mechanisms triggered by binding ephrin Bs to their specific receptors, a process called inside-outside signaling. The attachment of ephrin to its specific receptor triggers a specific sequence of events within the cell that activates the integrin receptor. In doing so, the integrin unfolds enabling it to bind with fibronectin. Additionally, Dr. Erickson explains the role of EphB and EDNRB2 in actin assembly. Since actin plays a significant role in cell motility, including creating meshwork at the leading edge of the cell and creating constricting contractile cables at the tail that pull the end of the cell forward, EphB and EDNRB2 may contribute to the motility of melanoblasts through actin organization. Dr. Erickson proposes that the EphB receptors may be linked to the Row family of GTPases involved in actin assembly, specifically Rack (actin leading edge assembly), CDC42 (assembles filopodia at leading edge), and Row (assembly of tail end contractile actin cables). Dr. Harris adds that experiments determining the relationship between the GTPases and EDNRB2 in dorsolateral migration had unclear results, although previous studies have shown that Eph is linked to GTPases through affixins and EDNRB2 is directly linked to Row GTPases. 7. Kimberly Johnson In the paper you hypothesized that the loss of repulsive molecules in the environment (repellants such as chondroitin-6-sulfate, PNA-binding molecules and F-spondin) may allow the DL to become permissive enough for melanoblasts to use this domain. Do you know yet of any substance or cell that is inhibiting these inhibitors at the time of melanoblast migration (or even a cell that acts as a sink for these molecules)? These inhibitory cues are not inhibited, but removed from the environment. It is still unknown whether these proteins are downregulated or cleaved. Dr. Erickson suggests that Adam 10, which is expressed in melanoblasts during DL migration, may be involved in the process. The Adam family proteins are multifunctional proteins important in cleaving proteins. Adam 10, specifically, cleaves cadherin which is critical for cell

adhesion. Thus, Adam 10 expressed in melanoblasts may also be cleaving inhibitory signals, clearing the way for melanoblast migration and creating a path of withdrawn extracellular molecules. As inhibitory chemical probes for Adam 10 were recently created, experiments determining changes in melanoblast DL migration due to Adam 10 inhibition are yet to be done. 8. Hanna Sherrill Do you think that there are cells in the pathway expressing receptors that help to sharpen the gradients as there are in primordial germ cell migration pathways, similar to what is observed in the interactions between CXCR7 and SDF1 during PGC migration in zebrafish? Dr. Harris states that the persistence of melanoblasts to travel within the dorsolateral pathway is not due to a sharp gradient. Instead, she proposes two other models for melanoblast persistence. Firstly, she suggests that persistence of melanoblasts to travel along the DL path is due to the path size and receptor upregulation. As melanoblasts move forward in the DL pathway, the c-kit receptor is upregulated. The interaction between this receptor and its ligand, steel factor, which is present in the ectoderm, is responsible for steering melanoblasts from the DL pathway to the ectoderm. Progression into the ectoderm is also driven by the small size of the DL path. Melanoblasts are forced forward as more melanoblasts try to enter the pathway. Secondly, Dr. Harris points out that melanoblast DL path persistence may result from cues from the emerging epidermis. 9. Rebecca Jacobson Some neural crest cells are pre-specified and some are not. Why do you suppose this is? What might the advantage be if for any NCC to be pre-determined? Is there a specific advantage to its ability to choose an appropriate pathway? Dr. Harris details the specification of neural crest cells at the vagal axial level. She generalizes that as you go from the head to the trunk during early development, you see increased complexity of the structures formed. The vagal axial level is located right between the head and the trunk and thus represents a midline of complexity in the early embryo. There are three waves of neural crest cell migration at the vagal axial level: the cardiac NCCs, the neural and glial NCCs, and finally the melanoblast NCCs. These neural crest cells are specified by their environment as they travel to their final destination. Cardiac NCCs travel through the dorsolateral pathway when very few inhibitors are present and thus require a low level of specification. Neural and glial neural crest cells migrate ventrally because inhibitors dissuade their entrance into the dorsolateral pathway. Since they do not need to reach their final destination through the dorsolateral pathway, neural and glial neural crest cells require a low degree of specification. Melanoblasts are the third wave of neural crest cells to migrate and must overcome the inhibitory cues in the dorsolateral pathway. Thus, they require a high degree of specification to allow them to travel dorsolaterally. Dr. Harris further explains that specification is advantageous because it helps neural crest cells arrive at their destination in the appropriate number. She uses the example of the

enteric system development. Neural crest cells migrate to the future location of the enteric system in the second wave of neural crest cell migration. There is a very specific window in which they can access the gateway to the gut. Specification allows neural crest cells to access this window. 11. Sarah Bashiruddin You say in your conclusion that you are interested in applying your findings in other model systems. Have you begun to look at applications in other model systems, maybe even in humans, medically or otherwise? How have you or are you planning to reconcile the deviation you see in chick melanoblast migration with those of mice, such as the varying role of c-kit between the two organisms? While different species demonstrate temporal differences in the molecular mechanisms that regulate melanoblast migration, this temporal difference is insignificant as they all still use the same mechanisms, like c-kit and slit1. C-Kit and slit1 are involved in the mouse system, the melanoblast invasion of ectoderm in chicks, and the survival of melanoblasts in chick. While c-kit is not required to direct melanoblasts dorsolaterally in chicks, it is still an important molecular mechanism maintained to direct melanoblast development. Previous research in melanoblast migration can be applied to mouse models and melanoma in humans. Dr. Harris will be starting her post-doc at NIH specifically investigating how genes important in melanoblast development play a role in melanoma. If gene candidates are identified, then researchers can apply what they understand for melanoblast development to the human disease of melanoma. 12. Michael Barresi Currently in your opinion what are the most pressing questions in the field of neural crest cell migration, and what steps is your lab taking to address these questions? The new front of neural crest cell research will be time lapse imaging. Time lapse imaging will allow researchers to actually place molecular cues into the context of cellular events through the power of observation. Time lapse imaging reveals that neural crest cells actually utilize a variety of means to migrate out of the neural tube. In summary, Dr. Erickson firmly believes that the future in the field of neural crest cell migration is the integration of visualization with molecular changes. Concluding Remarks One particular theme that continually arose in our conversation concerning melanoblast migration was the messiness or multiple paths that developmental biology takes to produce an organism. Melanoblasts present an elegant example of neural crest cell early specification, a specification that allows them to overcome the obstacles in the dorsolateral pathway; however, their migration is not as clearly defined as their specification initially suggests. Our conversation with Dr. Erickson and Dr. Harris provided a valuable dialogue to puzzle through and engage the complexities of melanoblast migration.