1. Ciprian Gheorghe MD PhD
Gene Expression in the Mouse Placenta: Developmental and Stress Responses
Ciprian Gheorghe MD PhD

3. Growth and Differentiation Contraction
Proposed Ca2+ -dependent and Ca2+ -independent pathways
Ca2+
Tyrosine Kinase R
a1-AR
PI3K
PKC
Ca2+
PLCb
DAG G
Src
PDK
SHC
Rho-A
IP3
GRB2
PAK K+
SOS
Rho Kinase
Ras
PKA SR
Ca2+
IP3R
MAPKKK(Raf)
CaM Kinase II
MAPKK (MEK)
MAPKK -P
Ca-CaM -P
CPI 17
CPI 17
Myosin
P42
P44
P42
P44
Erk
Erk -P
CaD -P
MLCP P-
MLCP
MLCK -P
MLCK
Transcription Factors
CaP -P
Actin + Myosin -P
Growth and Differentiation
Contraction

6. The Human Placenta
Ramsey, EM (1961)

7. Several gene defects can lead to disruption of this process:
Cross, JB (2001)
The mature murine placenta develops from the fusion at Embryonic day 8 of the allantois, with the extra embryonic chorion. The labyrinth develops through branching morphogenesis.
Several gene defects can lead to disruption of this process:
Giant cell differentiation
Chorioallantoic fusion.
Branching in the labyrinth
Nutrient transport in the labyrinth

8. Materials and Methods Breeding and tissue collection:
Eight-week old FVB/NJ male and female mice were obtained from the Jackson Laboratories (Bar Harbor, ME).
The mice were bred and the pregnant females were sacrificed at different time points during gestation.
The placentae were isolated under a dissection microscope.
Placentae from E10.5, E12.5, E15.5, and E17.5 were collected.
RNA was submitted to the UCI Microarray Core Facility for hybridization to the Affymetrix MG U74a microarray.

9. Results
To capture the evolving changes in gene expression in the developing mouse placenta we also compared expression levels between E10.5 and 12.5, E12.5 and 15.5 and finally 15.5 and 17.5.
We identified 622 probe sets that were differentially regulated between these time points corresponding to 599 individual genes.
We assigned functional classifications to the genes up- and down-regulated at each time point.

11. Hypoxia
Hypoxia is a well-known stressor in placental and fetal development
Contributes to pathologies such as pre-eclampsia, intra-uterine growth restriction.
In response to long-term exposure to high altitude, the ovine placenta undergoes significant morphological changes (Penninga & Longo, 1998).
Placental vasculature shows significant increases in capillary density, vessel tortuosity, and a decrease in diffusion distance (Krebs et.al, 1997).
Little is known about the mechanisms underlying these changes.
In the present study our objective is to help elucidate these mechanisms.

12. Materials and Methods Breeding and tissue collection:
Eight-week old FVB/NJ male and female mice were obtained from the Jackson Laboratories (Bar Harbor, ME).
The mice were bred and the pregnant females were subjected to hypoxia (10.5% Oxygen, equivalent to 0.5 atmosphere) from E15-E17.
Total RNA was submitted to the UCI Microarray Core Facility for hybridization to the Affymetrix Mouse E430A_v2 microarray .
Hybridization intensities were measured using the Affymetrix software.
Data was analyzed using the BRB Array Tools Software package.

13. Results
Hybridization intensities were measured for the 20,244 genes present on the microarray.
We analysed the data using the random variance method at a significance of p < (Wright, 2003).
171 genes were identified as being significantly regulated, and were selected for further analysis.
The genes were assigned to functional classes based on the GO database ( )
Significantly over-represented GO categories in the gene sets were analyzed using the Gene Ontology Tree Machine (

14. Genes of Interest Reactive Oxygen Species (ROS) metabolism.
Glutathione peroxidase
Ferrochelatase
NADPH Oxidase 4
Peroxiredoxin 2
eNOS
DNA methylation (epigenetic changes).
DNA methyltransferase 3b
Methyl CpG binding domain protein 1
RNA binding motif 3

15. Hypoxia
ROS
Glutathione Metabolism
DNA Methylation
Epigenetic changes
Long-term programming?

16. Why is it important
Provides potential mechanism of long term-programming. Barker hypothesis.
Epigenesis
Glutathione signaling. Not just ROS scavenging.

17. Conclusions
Hypoxic exposure triggers the following gene expression changes in the placenta:
Cell death
Heme metabolism
Fatty acid metabolism
Proteolysis and extra-cellular matrix remodeling
DNA methylation

18. Maternal Protein Deprivation
Maternal protein deprivation has been shown to have profound effects on fetal development.
Several studies have shown that caloric and particularly protein deprivation can have far reaching effects for the fetus, sometimes not manifesting itself until adulthood.
We exposed pregnant mice to a 50% reduction in protein content in the diet (10% protein by dry weight adjusted for caloric and nutrient content).
Mice were switched to the low protein diet from E10 to E17 in pregnancy.
Placentas were collected and gene expression measured as previously described.

19. Results
Hybridization intensities were measured for the 20,244 genes present on the microarray.
We analysed the data using the random variance method at a significance of p < (Wright, 2003).
171 genes were identified as being significantly regulated, and were selected for further analysis.
The genes were assigned to functional classes based on the GO database ( )
Significantly over-represented GO categories in the gene sets were analyzed using the Gene Ontology Tree Machine (

20. Genes of Interest Protein metabolism. Both up- and down-regulated.
Genes related to cell growth and maintenance, organogenesis, nucleotide metabolism, signal transduction and cell death were down-regulated.
Suggests that there is an overall depression in cell growth and metabolism in response to protein deprivation.
p53 pathway up-regulated
DNA/Histone acetylation/methylation

21. Protein Deprivation
p53 Pathway
G1 Arrest
Mitosis
Apoptosis
Metabolism
Cell Growth
DNA Methylation
Growth Restriction ?
Long-term changes

22. Placental stress responses
Hypoxia
Protein Deprivation
ROS metabolism
Cell growth
Proteolysis
Apoptosis
Cellular metabolism
Transcription
ECM remodeling
DNA Methylation
DNA metabolism
Metabolism
p53 Pathway
MAPKinase pathway
Placental stress responses

23. Acknowledgements Lawrence D. Longo MD Subburaman Mohan PhD
Steven M. Yellon PhD
Kerby Oberg MD PhD
Dennis Heck PhD
Ravi Goyal MD PhD