Progress 10/01/06 to 09/30/13

Outputs
OUTPUTS: The Silky fowl is a strain of chicken that is characterized by excessive pigmentation in the skin and internal organs. The goal of this project is to identify the genes and proteins responsible for the phenotype. To that end we proposed a genome-wide linkage analysis. In 2009 we were contacted by Chris Ashwell from the Department of Poultry Science at North Carolina State in Raleigh to tell us they had succeeded in cloning the fibromelanosis gene and have asked our help in doing the in situ hybridization assays. We have shared with them our expertise and reagents and they are now completing the study under the auspices of the Agricultural Experiment Station in North Carolina. The goal of the Ashwell lab is to identify genes of importance in the poultry industry. As I reported three years ago, we have shifted our research for the remained of the grant to more fundamental aspects of melanogenesis. During this time period (2006-2013) we have characterized the pathways that pigment cells take through the chicken embryo, we have determined the molecular mechanisms by which pigment cells find their way to the skin during embryogenesis, and we have determined the molecular pathways that control melanogenesis. It was been a very productive time for my lab and as I terminate this grant and also retire I am very pleased with the important contribution we have made to pigment cell migration in the chick embryo. Not only have these studies made fundamental contributions to morphogenesis during development, but have enhanced the poultry industry's understanding of melanogenesis in the deep tissues, which is an undesired trait. These discoveries have been shared through the peer-reviewed primary literature. PARTICIPANTS: Melissa Harris is now a postdoc at the NIH Aaron Thomas is now a postdoc at Utah State in the animal husbandry department Jon Ahlstrom is a postdoc at the U of Utah in a stem cell lab Bryan Kuo is a postdoc at St Jude Children's hospital Phil Spear is now a postdoc at Northwestern Their training allowed them to pursue these postdoc positions. TARGET AUDIENCES: The data on fibromelanosis were distributed to collaborators at North Carolina State. Since that time most of our data have been disseminated through seminars nationally and internationally and peer- reviewed publications. PROJECT MODIFICATIONS: As reported three years ago, our research is more focused on the basic biology of pigmentation in the chick since we shared our data on fibromelanosis with colleagues elsewhere and they are finishing the project. There was no point in both labs competing. We have continued to study control of migration of the neural crest, including pigment cells that are derived from the neural crest.

Impacts
For the past four years our research focus has changed from the original Hatch proposal since the cloning of the fibromelanosis gene was identified by another group. My graduate students in my lab made important contributions to our understanding of the basic development of pigment patterns in the chicken embryo. Melissa Harris identified two major receptor families (EDNRB, Eph receptor) that regulate the migration of pigment cells to the skin. These genes are of fundamental importance in developing pigment patterns. Aaron Thomas has elucidated the gene regulatory pathway for specification of the pigment cell. This is the first neural crest lineage whose gene regulation has been fully identified. More recently, Bryan Kuo has examined the migration of pigment cells and other neural crest derivatives at other axial levels in the chicken, which differ in their behavior compared to what Melissa and Aaron found. These data were published in Developmental Dynamics. Additional work in the lab involved studying the behavior of cells in the neural epithelium from which neural crest cells arise. Jon Ahlstrom used time-laspe imaging to capture neural crest cells as they undergo the epithelial/mesenchymal transition. Phil Spear also used time-lapse imaging to study another phenomenon in the neural epithelium called interkinetic nuclear migration. These studies were based on serendipitous observations as we were examining neural crest morphogenesis. There were two major technical outcomes. We have developed live cell confocal time-lapse imaging and developed techniques to knockdown gene expression in the chicken. These will be of fundamental importance in basic and applied research.

Publications

• Spear PC, Erickson CA. (2012) Apical movement during interkinetic nuclear migration is a two-step process. Dev Biol. 370(1):33-41.
• Ahlstrom, J.D. and Erickson, C.A. (2007) Organization of cells into higher ordered structures: the role of the epithelial-mesenchymal transformation in the generation and stabilization of embryonic tissues. In Principles of Tissue Engineering, 3rd Edition, Academic Press, pp. 67-79.
• Harris,M.L., Hall,R., and Erickson,C.A. (2008). Directing pathfinding along the dorsolateral path - the role of EDNRB2 and EphB2 in overcoming inhibition. Development 135, 4113-4122. PMID: 19004859
• Thomas,A.J. and Erickson,C.A. (2009). FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. Development 136, 1849-1858. PMCID: PMC2680109
• Ahlstrom,J.D. and Erickson,C.A. (2009). The neural crest epithelial-mesenchymal transition in 4D: a tail of multiple non-obligatory cellular mechanisms. Development 136, 1801-1812. PMCID: PMC2680107
• Kuo BR, Erickson CA. (2010). Regional differences in neural crest morphogenesis. Cell Adhesion and Migration 4, 1-19. Kuo BR, Erickson CA. (2011) Vagal neural crest cell migratory behavior: A transition between the cranial and trunk crest. Developmental Dynamics 240(9):2084-2100

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The Silky fowl is a strain of chicken that is characterized by excessive pigmentation in the skin and internal organs. The goal of this project is to identify the genes and proteins responsible for the phenotype. To that end we proposed a genome-wide linkage analysis. Last year we were contacted by Chris Ashwell from the Department of Poultry Science at North Carolina State in Raleigh to tell us they had succeeded in cloning the fibromelanosis gene and have asked our help in doing the in situ hybridization assays. We have shared with them our expertise and reagents and they are now completing the study under the auspices of the Agricultural Experiment Station in North Carolina. The goal of the Ashwell lab is to indentify genes of importance in the poultry industry. As I reported two years ago, we have shifted our research in this last year to more fundamental aspects of melanogenesis. PARTICIPANTS: Bryan Kuo is currently a post-doc at St. Jude's Hospital. Phil Spear is a graduate student who will be leaving for his postdoc in June 2012. TARGET AUDIENCES: The data on fibromelanosis were distributed to collaborators at North Carolina State. Since that time most of our data have been disseminated through seminars nationally and internationally and publications. Next month I will be giving a tribute to Ursula Abbott and her work at a memorial seminar in her honor on the UC Davis campus. PROJECT MODIFICATIONS: As reported a year ago, our research is more focused on the basic biology of pigmentation in the chick since we shared our data on fibromelanosis with colleagues elsewhere and they are finishing the project. There was no point in both labs competing. We have continued to study control of migration of the neural crest, including pigment cells that are derived from the neural crest.

Impacts
For the past three years our research focus has changed from the original Hatch proposal since the cloning of the fibromelanosis gene was identified by another group. Last year Bryan Kuo has examined the migration of pigment cells and other neural crest derivatives at the vagal level in the chicken, which differ in their behavior compared to what we have found at other axial levels. These data were finally published during this report period. Phil Spear, another graduate student in my group has been studying neurogenesis in the chicken and he has published a review article on his work on interkinetic nuclear migration. He has recently submitted his research data as well.

Publications

• Spear, P, Erickson, CA. 2012(In press) Interkinetic Nuclear Migration: A Mysterious Process in Search of a Function. Development, Growth and Differentiation

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The Silky fowl is a strain of chicken that is characterized by excessive pigmentation in the skin and internal organs. The goal of this project is to identify the genes and proteins responsible for the phenotype. To that end we proposed a genome-wide linkage analysis. Last year we were contacted by Chris Ashwell from the Department of Poultry Science at North Carolina State in Raleigh to tell us they had succeeded in cloning the fibromelanosis gene and have asked our help in doing the in situ hybridization assays. We have shared with them our expertise and reagents and they are now completing the study under the auspices of the Agricultural Experiment Station in North Carolina. The goal of the Ashwell lab is to indentify genes of importance in the poultry industry. As I reported two years ago, we have shifted our research in this last year to more fundamental aspects of melanogenesis. PARTICIPANTS: Bryan Kuo is currently a post-doc at St. Jude's Hospital. TARGET AUDIENCES: The data on fibromelanosis were distributed to collaborators at North Carolina State. Since that time most of our data have been disseminated through seminars nationally and internationally and publications. PROJECT MODIFICATIONS: As reported a year ago, our research is more focused on the basic biology of pigmentation in the chick since we shared our data on fibromelanosis with colleagues elsewhere and they are finishing the project. There was no point in both labs competing. We have continued to study control of migration of the neural crest, including pigment cells that are derived from the neural crest.

Impacts
For the past two years our research focus has changed from the original Hatch proposal since the cloning of the fibromelanosis gene was identified by another group. As reported last year two graduate students in my lab made important contributions to our understanding of the basic development of pigment patterns in the chicken embryo. Melissa Harris identified two major receptor families (EDNRB, Eph receptor) that regulate the migration of pigment cells to the skin. These genes are of fundamental importance in developing pigment patterns. Aaron Thomas has elucidated the gene regulatory pathway for specification of the pigment cell. This is the first neural crest lineage whose gene regulation has been fully identified. This year Bryan Kuo has examined the migration of pigment cells and other neural crest derivatives at other axial levels in the chicken, which differ in their behavior compared to what Melissa and Aaron found. These data have been submitted to Developmental Dynamics and are currently in revision.

Publications

• Kuo BR, Erickson CA. (2010). Regional differences in neural crest morphogenesis. Cell Adhesion and Migration 4, 1-19.
• Kuo BR, Erickson CA. Vagal neural crest cell migratory behavior: A transition between the cranial and trunk crest. Submitted to Developmental Dynamics 2010

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The Silky fowl is a strain of chicken that is characterized by excessive pigmentation in the skin and internal organs. The goal of this project is to identify the genes and proteins responsible for the phenotype. To that end we proposed a genome-wide linkage analysis. Last year we were contacted by Chris Ashwell from the Department of Poultry Science at North Carolina State in Raleigh to tell us they had succeeded in cloning the fibromelanosis gene and have asked our help in doing the in situ hybridization assays. We have shared with them our expertise and reagents and they are now completing the study under the auspices of the Agricultural Experiment Station in North Carolina. The goal of the Ashwell lab is to indentify genes of importance in the poultry industry. We have thus shifted our research in this last year to more fundamental aspects of melanogenesis. PARTICIPANTS: Melissa Harris was a graduate student in my lab. She recently began a postdoc at the NIH. Aaron Thomas was a recent graduate student in my lab who is now a postdoc at Utah State. TARGET AUDIENCES: In the last reporting period I disseminated knowledge to colleagues in two agricultural schools in North Carolina and Brazil. In this reporting period the knowledge has been distributed through publications and seminars given nationally and internationally. PROJECT MODIFICATIONS: As reported last year, another group did some of this work so we shared our data with them and provided them with the reagents to finish the project. My project switched emphasis to a more basic project to understand fundamental aspects of pigment cell differentiation and migration in the chicken and to determine genes that are of interest to the poultry industry.

Impacts
We changed our focus somewhat since the cloning of the fibromelanosis gene was identified by another group. Two graduate students in my lab made important contributions to our understanding of the basic development of pigment patterns in the chicken embryo. Melissa Harris identified two major receptor families (EDNRB, Eph receptor) that regulate the migration of pigment cells to the skin. These genes are of fundamental importance in developing pigment patterns. Aaron Thomas has elucidated the gene regulatory pathway for specification of the pigment cell. This is the first neural crest lineage whose gene regulation has been fully identified.

Publications

• Harris,M.L., Hall,R., and Erickson,C.A. (2008). Directing pathfinding along the dorsolateral path - the role of EDNRB2 and EphB2 in overcoming inhibition. Development 135, 4113-4122.
• Thomas,A.J. and Erickson,C.A. (2008). The making of a melanocyte: the specification of melanoblasts from the neural crest. Pigment Cell Melanoma Res. 21, 598-610.
• Kelsh,R.N., Harris,M.L., Colanesi,S., and Erickson,C.A. (2009). Stripes and belly-spots-A review of pigment cell morphogenesis in vertebrates. Semin. Cell Dev. Biol. 20, 90-104.
• Thomas,A.J. and Erickson,C.A. (2009). FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. Development 136, 1849-1858.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The Silky fowl is a strain of chicken that is characterized by excessive pigmentation in the skin and internal connective tissue. We have already identified the abnormal migratory behavior of the pigment cells that leads to internal pigmentation. The goal of this project is to identify the genes and proteins responsible for the phenotype. To that end we proposed to do a genome-wide linkage analysis and have collected nearly 100 offspring from heterozygous crosses. Recently we were contacted by Chris Ashwell from the Department of Poultry Science (Domestic Animal Genomics Laboratory) at North Carolina State in Raleigh, North Carolina to tell us they had succeeded in cloning the fibromelanosis gene and have asked our help in doing the in situ hybridization assays to determine the expression pattern of the gene. Since they may have indeed identified the gene we have stopped collecting offspring ourselves and are now sharing expertise, reagents and research time to help them complete their project. Thus we are disseminating knowledge to another research group in the Agricultural Experiment Station in North Carolina. The goal of the Ashwell lab is to identify genes of importance in the poultry industry PARTICIPANTS: Melissa Harris has recently completed her PhD and has stayed in my lab for an additional 9 months as a postdoc to complete work on the Silkie project. She will be leaving for a postdoc at the NIH in June 2009. Chris Aswell is a professor at UNC State in the Poultry Sciences department and studies genes that are critical to the poultry industry. Since he may have identified the fibromelanosis gene, we were happy to collaborate with him to determine the tissue expression of this gene in normal and Silkie embryos. Jeff Vitt is a talented undergraduate who has worked with Melissa on this project for over a year. Cloris Faraco is a long-time collaborator at the Federal University of Parana in Brazil. She has worked on Silkie chicken development with me for more than 15 years. TARGET AUDIENCES: One target for this work are basic and applied researchers, who have heard my talks at meetings. This dissemination of data has resulted directly in two collaborations, both of which are with individuals in agricultural departments in North Carolina and Brazil. PROJECT MODIFICATIONS: A major component of this project was to map the fibromelanosis gene. Since another group has apparently done this, we will now collaborate with them to finish the rest of the project (gene expression studies, mutation analysis etc.). If their gene turns out not to be correctly identified, we will return to the mapping project ourselves.

Impacts
Because the Silkie strain has abnormal and excessive migration of pigment cells into the connective tissues, as opposed to capture of melanoblasts just in the skin, we have examined what molecular components of the environment allow melanoblasts to migrate into inappropriate tissues. We have determined previously that pigment cells are attracted to ephrins in their environment and during this report period have shown that in the Silkie fowl, ephrins are expressed abnormally in the tissues that are normally not populated by pigment cells. During this report period Melissa Harris has also shown that when she knocks down the excessive ephrin expression by using siRNA constructs delivered by electroporation, that the pigment cells no longer migrate inappropriately. This work was done in collaboration with Cloris Faraco, who is a faculty member in the Federal Universityy in Parana, Brazil and works in the poultry sciences division of the Biology Department. This work is being prepared for submission. During this period Melissa also collected many more embryos for the gene mapping study, but as explained above, we are now collaborating with Chris Ashwell, who identified the gene before we did.

Publications

• No publications reported this period

Progress 01/01/07 to 12/31/07

Outputs
Elucidating the factors that are involved in developmental processes is sometimes best approached by studying subjects in which these processes have deviated from the normal. There is one naturally occurring example in birds, which which I have proposed to study, fibromelanosis in the Silkie fowl. In the adult Silkie fowl the fibromelanotic phenotype is characterized by pigmentation in both the skin and internal connective tissue (e.g. walls of blood vessels and mesenteries). However the actual genes and proteins that mitigate this deviant behavior are not known. I propose, through genome-wide linkage analysis, to identify the gene responsible for fibromelanosis in the Silkie fowl. Beginning May 2006 a heterozygous Silkie (Fibromelanosis) male was crossed to several Scaleless or Junglefowl hens. Fertilized eggs from these crosses were incubated until embryonic day 10-13, sacrificed, blood samples taken, and their phenotypes assessed. The offspring produced from the Junglefowl females were feathered and made identification of the fibromelanotic trait difficult. The Scaleless females, however produced offspring with very little feathering, and pigmentation in the dermis was highly visible. Four Scaleless females (#2528, #4878, #4884, #4893) were used for the remainder of progeny production. Through these families we have confirmed the mendelian inheritance of the Fibromelanosis trait. As of this date, we have established four Fibromelanosis/Scaleless families with a total of 61 offspring. Additionally, we have four female offspring from the original Silkie male that carry Fibromelanosis and will use these to produce F2 progeny. In the next year we will do the genetic mapping now that we have sufficient number of family members. We have also discovered by the candidate gene approach that ephrins, which are cell surface factors that stimulate pigment cell migration, are abberantly expressed in the Silkie embryo. When we perturb ephrins function, we inhibit migration of pigment cells in the Silkie fowl. This work has been submitted for publication.

Impacts
Pigmentation in the poultry industry is not a desired characteristic. Therefor the identification of this gene will be useful for breeding out pigmentation. Moreover our mapping study will provide a significant number of genetic loci that will be of use in the chicken genome community. We are collaborating with Mary Delaney to provide the linakage analysis.

Publications

• None 2007