View Project moody-affy-xenop-315103

Project Summary
Status:	Public
Publications:	1 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Xenopus	Array Design Detail	false
Organ/Tissue Type:	whole animal	Experiment Detail	true
Organ Region:	cochlea	Sample Detail	true
Cell Type:	embryonic tissues (palate)	Hybridization Detail	false
Study Type:	subclassification	Measurement Detail	false
Disease/Condition:	Mucopolysaccharidosis Type I and Wild type
Replicates:	4
Expected Samples:	16

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Investigator Contact Detail
Name	Dr. Sally A Moody
Institution:	George Washington University
Street Address:	Dept. Anatomy & Regen Biology
	2300 Eye Street, NW
City, State/Province:	Washington , DC
Zip/Postal Code:	20037
Country:	United States
Work Phone:	202-994-2878
Fax:	202-994-8885
E-mail:	samoody@gwu.edu

Proposal Detail

Grant:

R01NS23158-22

Status:

Public

Service Type:

Hybrization through Analysis

Study Relevance:

The goal of the current NS23158 funding is to understand how neural fate-stabilizing (NFS) genes function in order to provide fundamental knowledge about their potential roles in neural stem cell development. Neural fate-stabilization is characterized by the expansion of the neural plate shortly after the presumptive neural ectoderm has been induced by the repression BMP signaling. During this time period a number of early-expressed NFS genes are expressed, each of which expands the neural plate in gain-of-function assays. But, we do not know how these genes are related to one another or whether they are arranged in a linear gene pathway or multi-path hierarchy. A key aim of this grant is to elucidate the relationship of foxD5, an early-expressed NFS gene that we cloned, to other NFS-genes. We propose to greatly enhance this analysis by utilizing DNA microarray analyses to identify unsuspected and novel target genes.

Hypothesis:

FoxD5, a fork-head transcription factor which is expressed in the early neuroectoderm, is a key regulator of neural plate fate during expansion of the neural plate. Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of FoxD5, by the proposed microarray analyses. This information will allow us to study their function by gain- and loss-of function studies in the whole embryo.

Specific Aim:

What is the function of foxD5 in neural fate-stabilization, and how does it specifically relate to other genes in the early-expressed group? In the parent grant we proposed to study the relationship of early-expressed NFS genes to one that we cloned, FoxD5 (Sullivan et al 2001). FoxD5 expands the neural plate and holds it in an immature state. In the original grant, we proposed to study whether foxD5 activates or suppresses the expression of 6 known early-expressed NFS genes, using PCR and in situ hybridization of animal caps and whole embryos. Herein we propose to use DNA microarray technology to reveal a much broader spectrum of potential target genes.

Experimental Procedure and Design:

Animal cap (AC) explants are a naïve embryonic ectoderm that is removed from embryonic signaling centers. They allow one to perform gene induction assays in the absence of confounding growth factors, and thus are ideal for identifying downstream targets of transcription factors. In this experiment we will take a gain-of-function approach to identify which genes are induced/repressed by foxD5. FoxD5 mRNA (200pg) will be injected at the 2-cell stage, and embryos cultured until stage 8, at which time the zygotic genome begins transcription. AC explants will be cut from the embryos and cultured in Normal Amphibian’s Medium. When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be snap frozen in liquid nitrogen and RNA purified according to the Qiagen Rneasy Protect kit protocols. Our preliminary experiments indicate that 8-10ug of total RNA can be recovered with high purity from ~150 ACs with this method. As controls, RNA will be purified from uninjected animal caps derived from sibling embryos. If foxD5 represses NFS gene expression, it will not be detected in the above experiment because NFS genes are not expressed in AC explants in the absence of neural induction. Therefore, we will repeat the experiment but additionally treat the ACs (FoxD5-injected and sibling uninjected) at stage 8 with Noggin protein (R&D Systems) to induce neural ectoderm. For all four sample sets, RNA will be reverse transcribed, amplified and biotinylated using the NuGEN Ovation kit.

Quality Control Description:

(1) To test for the presence of known FoxD5 targets, and thus verify that the RNA sample will be representative of FoxD5 gene induction, an aliquot will be tested by standard PCR methods for the presence of sox2, an early zygotic neural ectoderm gene, according to standard methods used in our laboratory (Sullivan et al., 2001). (2) RNA purity will be tested by A260:A280 ratio. (3) RNA samples will be run on an Agilent Bioanalyzer to ensure RNA integrity.

Quality Control Types:

biological_replicate

Replicate Description:

Each sample will consist of ~150 pooled AC explants that are derived from 2 sets of parents. Control samples (uninjected, untreated; uninjected, Noggin-treated) will be derived from sibling embryos that are cultured in parallel. Each condition (uninjected control, FoxD5-injected, Noggin-treated, Noggin-treated+FoxD5 injected) will be repeated 5 times, providing a total of twenty samples.

Replicate Types:

biological_replicate

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.

Factor Name	Description	Factor Category
FoxD5 expression	In the experimental samples, FoxD5 ge...	genetic_modification

Project Samples

Samples associated with this project.

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Name	Description	Extracts
foxD5-noggin #1	foxD5-injected mRNA (100pg ...	1
foxD5-noggin #2	foxD5-injected mRNA (100pg ...	1
ctl-noggin #1	siblings of foxD5-noggin #1...	1
ctl-noggin #2	siblings of foxD5-noggin #2...	1
foxD5-noggin #3	foxD5-injected mRNA (100pg ...	1
ctl-noggin #3	siblings of foxD5-noggin #3...	1
foxD5-noggin #4	foxD5-injected mRNA (100pg ...	1
ctl-noggin #4	siblings of foxD5-noggin #4...	1
foxD5 #1	foxD5-injected mRNA (100pg ...	1
ctr #1	siblings of foxD5 #1; un-in...	1
foxD5 #2	foxD5-injected mRNA (100pg ...	1
ctr #2	siblings of foxD5 #2; un-in...	1
foxD5 #3	foxD5-injected mRNA (100pg ...	1
ctr #3	siblings of foxD5 #3; un-in...	1
foxD5 #4	foxD5-injected mRNA (100pg ...	1
ctr #4	siblings of foxD5 #4; un-in...	1

Project Hybridizations

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Name	Array	Labeled Extract
Xenopus_laevis_1_hyb	Xenopus_laevis_1	cDNA labeling
Xenopus_laevis_2_hyb	Xenopus_laevis_2	ctr #1_e1_le1
Xenopus_laevis_3_hyb	Xenopus_laevis_3	foxD5 #2_e1_le1
Xenopus_laevis_4_hyb	Xenopus_laevis_4	ctr #2_e1_le1
Xenopus_laevis_5_hyb	Xenopus_laevis_5	foxD5 #3_e1_le1
Xenopus_laevis_6_hyb	Xenopus_laevis_6	ctr #3_e1_le1
Xenopus_laevis_7_hyb	Xenopus_laevis_7	foxD5 #4_e1_le1
Xenopus_laevis_8_hyb	Xenopus_laevis_8	ctr #4_e1_le1
Xenopus_laevis_9_hyb	Xenopus_laevis_9	foxD5-noggin_e1_le1
Xenopus_laevis_10_hyb	Xenopus_laevis_10	foxD5-noggin #2_e1_le1
Xenopus_laevis_11_hyb	Xenopus_laevis_11	ctl-noggin #1_e1_le1
Xenopus_laevis_12_hyb	Xenopus_laevis_12	ctl-noggin #2_e1_le1
Xenopus_laevis_13_hyb	Xenopus_laevis_13	foxD5-noggin #3_e1_le1
Xenopus_laevis_14_hyb	Xenopus_laevis_14	ctl-noggin #3_e1_le1
Xenopus_laevis_15_hyb	Xenopus_laevis_15	foxD5-noggin #4_e1_le1
Xenopus_laevis_16_hyb	Xenopus_laevis_16	ctl-noggin #4_e1_le1

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