GLOBAL SURVEY OF SNAIL2 TARGET GENES DURING AVIAN GROWTH AND DEVELOPMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0220912
• Project No.: MD-ANSC-1124
• Project Start Date: Nov 20, 2009
• Project End Date: Nov 19, 2014

Project Director
Taneyhill, LI, A.

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
Animal and Avian Sciences

Non Technical Summary
A lack of data currently exists with respect to Snail target genes in the chicken embryo. These target genes play a critical role in transforming premigratory neural crest cells into migratory neural crest cells that ultimately create the chicken embryo, allowing it to develop into an adult organism. Through the use of traditional embryology and cutting-edge molecular biology, biochemical and genomics assays, we will catalog genes required to transform a chicken embryo into an adult. Obtaining this information is absolutely essential in order to allow us to address issues relating to chicken growth and development, including the ability to develop a more robust chicken based on our knowledge of such things as susceptibility to disease, nutritional requirements, behavior and breeding preferences. Collectively, these insights from basic research will enable us to move more easily into applied research, leading to a change in knowledge (disseminated in scientific publications), actions (application and use of our research results within the poultry industry), and conditions (higher productivity in poultry products).

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3210	1050	50%
305	3210	1050	50%

Knowledge Area
305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3210 - Egg-type chicken, live animal;

Field Of Science
1050 - Developmental biology;

Keywords

chicken

embryo

neural crest

snail2

gene regulation

chip

chip-seq

genomics

embryology

Goals / Objectives
The neural crest, a population of migratory cells derived from the future central nervous system in the developing chicken embryo, gives rise to a diverse range of cell types, including most of the peripheral nervous system, melanocytes, the craniofacial skeleton, and parts of the heart. As such, the proper formation of neural crest cells is critical for the overall growth and development of the chicken embryo into the adult organism. Initially existing as adherent epithelial cells in the embryonic dorsal neural tube (the premigratory neural crest), these cells undergo an epithelial-to-mesenchymal transition (EMT), characterized by the loss of intercellular contacts and the generation of motile, mesenchymal neural crest cells. The Snail family of transcriptional repressors is known to play key roles during EMTs underlying normal developmental and aberrant processes, the latter exemplified by the metastasis of cancer cells. Although some Snail targets have been identified in other systems, those targets of Snail2 mediating neural crest EMT/migration remained elusive, primarily due to experimental limitations that have now been resolved by our prior work. Our long-term goal is to establish the role of Snail2, and the genes Snail2 regulates, in chick neural crest development. The objective of this HATCH project is three-fold: 1) Develop and execute an innovative research methodology designed to identify novel targets of Snail2 in a global survey of the chick genome (Years 1 and 2); 2) Biochemically verify these targets using in vivo assays in the chick (Year 3), and 3) Establish the functional role of Snail2 targets in the development of the chick embryo (Years 4 and 5). Our central hypothesis is that genes regulated by Snail2 are likely to play a critical role in transforming a precursor epithelial cell in the dorsal neural tube into a migratory neural crest cell. The rationale is that the discovery of Snail2 target genes will shed light on both the processes regulating neural crest formation and the fundamental molecular mechanisms controlling EMTs that are required for appropriate animal growth and development. The expected outputs from this project include conducting and analyzing experimental data, participation in national and international meetings to present our findings, the publication of our data, and the establishment of a gene database to facilitate the dissemination of our findings after publication.

Project Methods
We propose to perform a series of experiments to address the objectives of this HATCH project. We will first use a new sequencing approach (Illumina-based sequencing), coupled with chromatin immunoprecipitation (ChIP), to catalog possible targets of Snail2 regulation during neural crest development (ChIP-Seq). Enrichment of DNA sequences associated with Snail2 from chick premigratory neural crest is achieved through the use of ChIP with a Snail2 antibody. The result is a series of short sequence reads that can be aligned to a reference genome (chick), thereby allowing for the identification of potential binding sites for Snail2. Importantly, ChIP-Seq has been successfully employed to identify transcription factor binding sites within the genomes of multiple species from cultured cell lines as well as in various animal models. Thus, our work will be pioneering through the adaptation of ChIP-Seq to an outstanding embryological system (chick) in order to define gene expression changes during neural crest cell EMT/migration that lead to the formation of the chicken. Premigratory neural crest tissue will be dissected from the midbrain regions of appropriately staged chick embryos (2-7 somite stage) using tungsten needles and pooled to collect starting material for the assay. At these stages of chick development, Snail2 is expressed exclusively in the premigratory and migratory cranial neural crest and is absent from other tissues, such as the mesoderm. Using a ChIP method previously established by the PI for the chick embryo, we will isolate Snail2-bound chromatin from dissected tissue using antibodies specific to Snail2. Mouse IgG and RNA polymerase II antibodies will be employed in an identical reaction as negative and positive controls, respectively. Identified target sequences will be mapped back to the chick genome, and the presence of multiple sequencing reads for a particular genomic site will indicate that this site is likely to be bound by Snail2 in vivo. Importantly, these sites will lie within regions up- and/or downstream of putative Snail2 targets. Candidate target sequences will be verified biochemically through independent ChIP assays in the chick embryo coupled with quantitative PCR (QPCR). Upon confirmation of Snail2 interactions at these loci, the genes to which these sequences are linked will be knocked-down or overexpressed in chick using morpholino antisense oligonucleotides and gene overexpression constructs, respectively, to determine the effects of gene perturbation on neural crest and overall chick growth and development. These latter assays will be evaluated using in situ hybridization, immunostaining and/or QPCR. Key milestones in this project will include the design and successful execution of ChIP-Seq in the chick, mapping of sequencing reads to the chicken genome to identify Snail2 target genes, validation of identified sequences using independent assays, and the functional characterization of target genes through in vivo and in vitro experiments. Taken together, these efforts will provide experiential learning opportunities and instruction for all involved in the laboratory.

Progress 11/20/09 to 11/19/14

Outputs
Target Audience: The target audience was the general developmental biology community as well as new University of Maryland undergraduates and local high school students for whom experiential learning opportunities were provided Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Various activities for this projected included the following: 1) conducting experiments with a postdoc and high school, graduate and undergraduate students; 2) mentoring the above personnel; and 3) teaching personnel how to do embryology and carry out biochemical and molecular biology experiments. How have the results been disseminated to communities of interest? Results have been disseminated through 1) peer-review publications, 2) participation by the PI and lab personnel at the regional Society for Developmental Biology meetings, 3) talks given by the PI at different university seminar series, and 4) dissemination of materials at meetings through poster and oral presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue carrying out molecular, cell, and embryology assays to address how these Wnt and Snail2 target genes regulated neural crest and placode cells during the formation of the cranial ganglia.

Impacts
What was accomplished under these goals? This project led to a change in knowledge by allowing us to demonstrate that the Snail2 target aN-catenin is expressed later by migratory neural crest cells as they interact with placode cells to form the cranial trigeminal ganglia. Additional molecules expressed initially by neural crest cells were also investigated, including the Wnt target Annexin A6. We found that this protein is lost by early migratory neural crest cells but is later expressed in placode cells during their induction, migration and interaction with neural crest cells during trigeminal ganglia assembly. We also showed that aN-catenin works through Cadherin-7 to promote productive interactions with placode cells, while Annexin A6 may serve as a cytoskeletal scaffolding molecule and direct changes in actin to permit placode cell motility and associations with neural crest cells.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Shah A and Taneyhill LA. Functional role of the Wnt target Annexin A6 in trigeminal ganglia formation. Society for Developmental Biology 73rd Annual Meeting. July 2014. University of Washington, Seattle
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Schiffmacher AT, Padmanabhan R, Jhingory S, and Taneyhill LA. 2014. Cadherin-6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural crest. Mol Biol Cell 25: 41-54. (Epub ahead 2013 Nov 6).
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Fairchild CL, Conway, JP, Schiffmacher AT, Taneyhill LA, and Gammill LS. 2014. FoxD3 regulates cranial neural crest EMT via downregulation of Tetraspanin18 independent of its functions during neural crest formation. Mech Dev. 132: 1-12.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wu CY, Hooper RM, Han K, and Taneyhill LA. 2014. Migratory neural crest cell ?N-catenin impacts chick trigeminal ganglia formation. Dev Biol. 392: 295-307.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience was the general developmental biology community as well as new University of Maryland undergraduates and local high school students for whom experiential learning opportunities were provided. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Various activities for this projected included the following: 1) conducting experiments with a postdoc and high school, graduate and undergraduate students; 2) mentoring the above personnel; and 3) teaching personnel how to do embryology and carry out biochemical and molecular biology experiments. How have the results been disseminated to communities of interest? Results have been disseminated through 1) peer-review publications, 2) participation by the PI and lab personnel at the regional Society for Developmental Biology meetings, 3) talks given by the PI at different university seminar series, and 4) dissemination of materials at meetings through poster and oral presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue carrying out molecular, cell, and embryology assays to address how target genes are regulated by Snail2, and what their function is in premigratory neural crest cells during EMT. We will also investigate how these target genes function in migratory neural crest cells and placode cells during the formation of neural crest derivatives.

Impacts
What was accomplished under these goals? This project led to a change in knowledge by allowing us to demonstrate that Annexin A6, a gene whose protein product functions at cellular membranes and controls cell adhesion and migration in vitro, plays an important role both early neural crest migration in the formation of migratory placode cells during trigeminal ganglia formation in vivo. Our data show that Annexin A6 is a Wnt target that may be regulated through Snail2. Annexin A6 is expressed by premigratory and early migrating neural crest cells, after which it is down-regulated and only observed in placode cells, both in the surface ectoderm and as they ingress and intermingle with neural crest cells to form the trigeminal ganglia. Functional assays revealed the Annexin A6 knock-down and overexpression in either migratory neural crest cells or placode cells affects trigeminal ganglion assembly. We are now investigating whether Annexin A6 modulates cadherin levels in both neural crest and placode cells.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Shah A and Taneyhill LA. Functional role of the Wnt target Annexin A6 in trigeminal ganglia formation. Society for Developmental Biology Mid-Atlantic Regional Meeting. April 19-21, 2013. College of William and Mary, Williamsburg, VA, USA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Hooper RM and Taneyhill LA. The role of the adherens junction protein aN-catenin in cranial neural crest cell differentiation. Society for Developmental Biology 71st Annual Meeting, July 19-23, 2012. Montreal, Canada.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Wu C-Y and Taneyhill LA. Annexin A6 modulates cranial neural crest cell migration. Society for Developmental Biology 71st Annual Meeting, July 19-23, 2012. Montreal, Canada.

Progress 01/01/12 to 09/30/12

Outputs
Target Audience: The target audience is the general developmental biology community. Experiential learning opportunities were also provided for undergraduates who provides some assistance to the above personnel. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Various activities for this projected included the following: 1) conducting experiments with postdocs, a technician, and high school, graduate and undergraduate students; 2) mentoring the above personnel; and 3) teaching personnel how to do embryology and carry out biochemical and molecular biology experiments. How have the results been disseminated to communities of interest? Results have been disseminated through 1) peer-review publications, 2) participation by the PI and lab personnel at the regional and annual Society for Developmental Biology meetings, 3) talks given by the PI at different university seminar series, and 4) dissemination of materials at meetings through poster and oral presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue carrying out molecular, cell, and embryology assays to address how target genes are regulated by Snail2, and what their function is in premigratory neural crest cells, prior to and during EMT.

Impacts
What was accomplished under these goals? This project led to a change in knowledge by allowing us to demonstrate that Annexin A6, a gene whose protein product functions at cellular membranes and controls cell adhesion and migration in vitro, is regulated by Wnt and plays an important role in neural crest emigration in vivo. Functional assays revealed the Annexin A6 knock-down and overexpression decreases or increases the number of migratory neural crest cells, respectively. This occurs during the process of neural crest cell emigration (or EMT) through the retention or premature down-regulation of premigratory neural crest cell cadherins, which normally function to hold these cells together. These results are the first to describe a role for Annexin proteins in the neural crest. We are now investigating how Annexin A6 modulates neural crest cell cadherin levels. In addition, we discovered that the transmembrane tight junction protein claudin-1 influences neural crest cell emigration. Claudin-1 depletion or overexpression enhances or impedes neural crest cell emigration, respectively, and does this independently of affecting adherens junctions or the neural tube basal lamina. We will next examine the temporal order of how these proteins are down-regulated during neural crest cell EMT. Other comments: The Claudin-1 experiments were carried out by an HHMI undergraduate, Ms. Theresa Neiderer, who is now at med school at the University of Buffalo.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2012 Citation: Fishwick KJ, Neiderer TE, Jhingory S, Bronner ME, and Taneyhill LA. The tight junction protein claudin-1 influences cranial neural crest cell emigration. Mech Dev. 2012; 129: 273-283.
• Type: Journal Articles Status: Accepted Year Published: 2012 Citation: Wu C-Y and Taneyhill LA. Annexin A6 modulates chick cranial neural crest cell emigration. PLoS One 2012; 7:e44903.

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

Outputs
OUTPUTS: Various activities for this projected included the following: 1) conducting experiments with postdocs, a technician, and high school, graduate and undergraduate students; 2) mentoring the above personnel; 3) teaching personnel how to do embryology and carry out biochemical and molecular biology experiments; 4) participation by the PI and lab personnel at the regional and annual Society for Developmental Biology meetings; 5) dissemination of materials at meetings occurred through poster and oral presentations. PARTICIPANTS: Participants in this project included the following: 1) PI: Lisa Taneyhill, 2) Technician: Sharon Jhingory, and 3) Postdoc: Chyong-Yi Wu. The postdoc and tech both attended the national Society for Developmental Biology meeting to gain more exposure on developmental biology in a variety of systems as well as career options in this field. TARGET AUDIENCES: The target audience was the general developmental biology community. Experiential learning opportunities were also provided for undergraduates who provided some assistance to the above personnel. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project led to a change in knowledge by allowing us to demonstrate that cingulin, a gene whose protein product functions at tight junctions to hold cells together, is regulated by Snail2 and plays an important role in neural crest emigration. Functional assays revealed the cingulin knock-down and overexpression give a similar phenotype (increase in the number of migratory neural crest cells), but do so through very different molecular mechanisms. These results provided insight into how the tight regulation of one molecule is absolutely crucial during neural crest cell development. These results then led us to a change in action, as we are now investigating in greater detail how changes in different molecules affect the premigratory neural crest cell population, which in turn affects the number of migratory neural crest cells. In addition, we are also looking at the interplay between cell adhesion molecules and the basement membrane/basal lamina, a region overlying the neural tube that must be degraded/removed prior to neural crest cell emigration.

Publications

• Wu C-Y, Jhingory S, and Taneyhill LA. 2011. The tight junction scaffolding protein cingulin regulates neural crest cell migration. Dev Dyn 240: 2309-2323.