GENE EXPRESSION DURING GONAD DEVELOPMENT

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0198615
• Project Start Date: Oct 1, 2003
• Project End Date: Jan 31, 2005
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
VETERINARY BIOSCIENCES

Non Technical Summary
Recently, gene expression profiling has emerged as a powerful tool to identify a large number of differentially expressed genes, and ultimately whole genetic programs when comparing various samples. I hope to decipher the signaling mechanisms initiated by gene expression cascades that result in sex determination, gonad formation, and healthy germ cells.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3999	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1020 - Physiology;

Keywords

gene expression

gonads

organogenesis

animal physiology

reproductive performance

testes

ovaries

animal development

differentiation

metabolic pathways

promoters (genetics)

binding sites

males

females

phenotypes

signal transduction

sex determination

germ cells

birth defects

infertility

Goals / Objectives
The molecular events underlying the transformation of the indifferent mammalian gonad into a testis or ovary are largely a mystery. While a handful of genes have been identified as present during determination or differentiation of gonadal tissue, very few of these have actually been linked to a specific pathway. Limited examples such as SF-1 binding sites on the Dax-1 promoter, or SRY working through Sox9 for testis determination, illustrate that clear networking in gonadal development remains to be defined. Thus, the goal of this research is to begin to identify a network of genes that are responsible for the determination of gonadal tissue, and the differentiation into a male or female gonad. In particular, we hypothesize that the pursuit of both male and female phenotypes are active processes that depend on expression or suppression of specific interactive pathways

Project Methods
When comparing specific developmental time points between males and females, over 36,000 genes and ESTs can be screened to discover new genes that will begin to identify unique pathways and will lay the foundation for further understanding of the events that dictate male or female gonad development. Thus, we have performed large-scale gene expression profiling studies of developing gonad transcriptomes using Affymetrix GeneChips. Subtractive layers of gene expression comparisons were performed by analyzing male versus female gonadal ridges at four specific time points ranging form E12 to E13.5. Currently, we are focusing on several genes that increase at least three-fold in females while remaining unchanged or absent in males. The goal of my research program is to utilize the Affymetrix Gene Chip data to discover new and existing genes that are important for male and female gonad development. I intend to ascertain their relative importance by explant culture and electroporation as described above and by making conditional knock-out or knock-in mice. From here, I intend to uncover upstream factors by evaluating 5' regulatory sequences and discover downstream target genes by several mechanisms including chromatic immunoprecipitation cloning (ChIP cloning).

Progress 10/01/03 to 01/31/05

Outputs
The Affymetrix genechip data has been verified by comparing expression profiles from genechip data to patterns of expression that have been published. We assessed over 20 genes and found 100% agreement in the patterns between the genechip and published sources. These included genes relevant to gonad development, for example, we verified males specific factors Desert hedghog, Sox 9, and Mullerian Inhibiting Substance (MIS); female specific factors Wnt4, Xist, and Follistatin; and steroidogenic pathway genes StAR, Cyp 11a1 (p450 Side Chain Cleavage) and HSD3beta-1. Furthermore, we used real time quantitative PCR to validate four genes novel to gonad development. These include Irx3, G protein subunit gamma 13, Septin 7, and IGFBP2, all of which displayed a sexually dimorphic expression profile in both genechip data and PCR validation. These results suggest the validity of our genechip data and allow us to proceed with assessing additional genes with greater confidence.We have successfully developed a technique to assess promoter activity in embryonic gonad explant cultures. This is being used to assess the sexually dimorphic nature of expression of genes identified in our Affymetrix gene chip profiling experiment. These experiments will be critical in understanding regulation of genes that develop sexually dimorphic expression patterns as the embryonic gonad transitions from being a bipotential organ to one of male or female specific patterning.

Impacts
The Affymetrix Genechip data describes expression patterns of genes that are relevant during the time of sex determination of the mouse. Knowledge of the genetic pathways that drive the transition of a bipotential organ to development of either a testis or ovary is critical in our understanding of several important fields including basic development which will help us to understand causes of birth defects associated with sexual identity, causes of infertility, and factors that may lead to the development of testicular or ovarian cancer later in life.

Publications

• Jorgensen JS, Gao L 2005 Irx3 Is Differentially Up-regulated in Female Gonads During Sex Determination Gene Expr Patterns 5/6:756-762.

Progress 10/01/03 to 09/30/04

Outputs
The Affymetrix genechip data has been verified by comparing expression profiles from genechip data to patterns of expression that have been published. We assessed over 20 genes and found 100% agreement in the patterns between the genechip and published sources. These included genes relevant to gonad development, for example, we verified males specific factors Desert hedghog, Sox 9, and Mullerian Inhibiting Substance (MIS); female specific factors Wnt4, Xist, and Follistatin; and steroidogenic pathway genes StAR, Cyp 11a1 (p450 Side Chain Cleavage) and HSD3beta-1. Furthermore, we used real time quantitative PCR to validate four genes novel to gonad development. These include Irx3, G protein subunit gamma 13, Septin 7, and IGFBP2, all of which displayed a sexually dimorphic expression profile in both genechip data and PCR validation. These results suggest the validity of our genechip data and allow us to proceed with assessing additional genes with greater confidence. We have successfully developed a technique to assess promoter activity in embryonic gonad explant cultures. This is being used to assess the sexually dimorphic nature of expression of genes identified in our Affymetrix gene chip profiling experiment. These experiments will be critical in understanding regulation of genes that develop sexually dimorphic expression patterns as the embryonic gonad transitions from being a bipotential organ to one of male or female specific patterning.

Impacts
The Affymetrix Genechip data describes expression patterns of genes that are relevant during the time of sex determination of the mouse. Knowledge of the genetic pathways that drive the transition of a bipotential organ to development of either a testis or ovary is critical in our understanding of several important fields including basic development (which will help us to understand causes of birth defects associated with sexual identity) causes of infertility, and factors that may lead to the development of testicular or ovarian cancer later in life.

Publications

• No publications reported this period