GenomeWide Studies of Early Embryo Development

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Genome-Wide Studies of Early Embryo Development

• Rong Kong, Nell Keith, Rohith Reddy
• BIOE598SZ
• 10/11/2006

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Genome-Wide Studies of Embryo Development

• Questions
• Which genes are important in early embryo
  development?
• What are the expression patterns of these genes
  over time?
• Does in-vitro culture affect embryo development?
• Methods
• Genome-wide studied on preimplantation mouse
  embryo
• In vivo and in vitro

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In vivo and In vitro

• In vivo
• refer to experimentation done in or on the living
  tissue of a living organism
• e.g. animal testing and clinical trials
• In vitro
• refer to the technique of performing a given
  experiment in a test tube, or, generally, in a
  controlled environment outside a living organism
• e.g. In vitro fertilization

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Some Ways to Identify Important Genes in
Development

• Molecular genetics
• Phenotype-based
• Examples gene knockout, over expression
• Successful in identifying gene functions
• Limited in early embryo
• Genome-scale expression analysis
• Expression-based
• More gene discovery
• Examples EST sequencing, suppression subtractive
  hybridization, microarray

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Microarray Study

• Pros
• Expression-based, not phenotype-based
• High throughput compared with other methods
• Overall picture
• Quantitative results

• Cons
• Difficult to obtain sufficient RNA in early
  embryo
• Require linear amplification

Microarray-based gene expression studies had been
done in C. elegans and Drosophila.
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Stages of Early Development
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Oogenesis Germinal Vesicle (GV) and metaphase II
oocyte
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Fertilization Zygote
In animals, fertilization is the process that a
sperm fuses with an ovum, which eventually leads
to the development of an embryo.
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Zygotic Genome Activation (ZGA)

• The onset of new transcription
• Degradation of maternal transcripts
• ZGA happens during the transition from oocyte to
  embryo

• Examples of maternal transcripts
• zp1-3 zona pellucida glycoproteins
• tPA oocyte maturation in meiosis II

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Cleavage 2-cell, 4-cell, 8-cell, Morula stages
Cleavage is the division of cells in the early
embryo. The zygotes undergo rapid cell cycles
with no significant growth, producing a cluster
of cells the same size as the original zygote.
1232 cells
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• Compaction

• Adhesion molecules
• ZO-1, Cx43
• integrin, Cadherin

A process that individual cells begin to adhere
tightly
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Blastocyst
A strucuture consisting of an inner cell mass (or
embryoblast) and an outer cell mass (or
trophoblast).
ICM gt embryonic stem cells
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Mid-preimplantation gene activation (MGA)

• Another wave of gene activation
• Companied by the dramatic morphological changes
• From compaction to morulae and blastocyst stages

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Implantation
A process in which blastocyst adheres to and
enters into the uterine wall.
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Some Signal Pathways
Notch pathway
Wnt pathway
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References

• Developmental Biology (7th Edition). Gilbert SF.
• Principles of Development. Wolpert L.
• Molecular Biology of the Cell. 4th Edition.
  Alberts B, et al.
• Wikipedia

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A Genome-Wide Study of Gene Activity Reveals
Developmental Signaling Pathways in the
Preimplantation Mouse Embryo

• Wang et. al., Developmental Cell, 2004

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Overview

• Examine variation in overall gene expression
  levels during 12 stages of embryogenesis
• In vivo study
• Relate expression profile of specific genes to
  biological function
• Explore conserved regulators of cell fate in
  embryo development
• Wnt pathway
• Notch pathway

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Gene Expression Consistency in Each Individual
Embryo Stage

• Embryos staged based on morphological criteria
• Transcription profiles examined with Affymetrix
  Mouse
• GeneChip
• Pearson correlation examined to assess
  similarity between
• profiles

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Gene Expression Variation in Consecutive Embryo
Stages

• Pearson correlation between consecutive embryo
  development stages
• Steeper curve indicates greater change in
  transcript composition

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3-Fold Change in Gene Expression Between Embryo
Stages

• Significant degradation of maternal mRNA during
  meiotic maturation
• Changes in gene expression less pronounced after
  compaction

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Relative Levels of Transcription

• Determined percentage of genes receiving present
  calls (P call )
• P call decrease during meiotic maturation due
  to maternal mRNA degradation
• After compaction and embryo layer development
  changes in P call are difficult to detect

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Hierarchical Clustering

• 12 stages divided into two groups
• Change in gene expression reflects morphological
  development of embryo

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Regulation of Gene Expression at Various Stages
during Preimplantation Development

• Peak expression level is set as 100 and the
  expression levels at other time points are
  normalized to percentage of peak level (peak)
• Expression patterns of several maternal
  transcripts during the transition from oocyte to
  embryo Zp1-3, mos, tPA, and SLBP

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Regulation of Gene Expression at Various Stages
during Preimplantation Development

• Expression curves of ZO-1 and Cx43
• cell adhesion genes
• function during and after compaction
• Possible unknown function of ZO-1?

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Regulation of Gene Expression at Various Stages
during Preimplantation Development

• Expression curves of Eomes and Crtr-1, two genes
  with lineage-specific functions.
• Encode transcription factors that regulate cell
  fate and pluripotency in the blastocyst.

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Maternal Transcription of Genes Implicated in
Setting Up Polarity

• mos and tPA were used as landmarks to focus
  clustering
• 57-gene cluster of maternal transcripts that are
  most abundant during oocyte maturation
• Relative expression levels
• Blue low level
• Red high level
• Colored coded genes involved in embryo
  development
• Blue resume cell cycle
• Red establish cell polarity

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Maternal Transcription of Genes Implicated in
Setting Up Polarity

• B Average trend of expression of the cluster
• C Immunofluorescent staining of ovarian sections
  confirms expression of the DAG protein in GV
  stage oocytes

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Comparison of Temporal Expression Patterns of
Oct4 Cofactors and Targets

• Pou5f1 encodes the transcription factor Oct4
• Sox2 is a cofactor of Oct4
• HMG2 is possible cofactor for Oct4

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Wnt and Notch Pathways

• Work in conjunction
• Wnt promotes division
• Notch promotes differentiation

• Stages of activity
• oocyte to embryo
• preceding implantation

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The Wnt Signaling Pathway Is Active at the
Blastocyst Stage
A-C Inner Cell Mass D-F Trophectoderm

• RNA encoding ß-catenin-GFP injected at 16-cell
  stage and subcellular location examined in
  blastocyst
• Conclusions
• Wnt pathway active first in ICM
• Wnt pathway initiated before implantation

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Gene expression in preimplantation embryos from
In-vitro study

• Paper by Shunping Wang et al., Reproductive
  Biomedicine Online, 2005

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Overview

• Hierarchical clustering of gene expression at
  various developmental stages
• Extent of Up and Down regulation
• 2- to 4-cell gene ontology
• Comparisons of gene expression patterns between
  cultured (in-vitro) embryos and in-vivo embryos
• Conclusion

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Hierarchical clustering of gene expression at
various developmental stages

• Results from Hierarchical clustering
• Clustering based on similarity of gene patterns
• Distance Pearsons correlation coefficient,
  centroid linkage method used
• Note the closeness and some intermixing of the
  8-cell and compacting embryos, which indicates
  similar expression profiles

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Extent of Up and Down regulation

• Figure shows the number of genes up and
  down-regulated
• The threshold was set as 2-fold change
• Note significant numbers of genes were up- and
  down regulated when embryos developed from 1- to
  2-cell stage, while almost no change was observed
  between 8-cell and compacting embryos

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Extent of Up and Down regulation

• Developmental profile of gene regulation for both
  in-vitro and in-vivo.
• Y-axis represents the number of genes up- or
  down-regulated, using 2-foId change as threshold.
  
• Note the similar trends of up-regulated genes for
  both in vivo and in vitro., except the stage
  between 2- and 4-cell stage.

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2- to 4-cell gene ontology

• A significant number of genes (693 genes) were
  observed between in-vitro and in-vivo embryos in
  terms of up-regulated gene numbers when they
  developed from 2- to 4-cell
• Pie chart shows the gene ontology (the molecular
  function of gene products) analysis of these 693
  genes
• Note that metabolism-related genes account tor
  66 of the total genes

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Comparisons of gene expression patterns between
cultured embryos and in-vivo embryos

• E2F-1 is a transcription factor that plays an
  important role in apoptosis (programmed cell
  death) pathway in early embryos.
• E2F-1 expression levels increased at 2-cell stage
  and decreases after 4-cell stage
• In-vivo and in-vitro plots are remarkably similar
• Caspase is a protease (helps breakdown proteins)
  that is involved in initiating cellular events of
  apoptosis in preimplantation stage embryos
• Very little is known about where and when these
  genes are expressed.
• Low till 2-cell stage and increases till
  compacting stage

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Comparisons of gene expression patterns between
cultured (in-vitro) embryos and in-vivo embryos

• Jak2 (Janus kinase 2) is involved in cytokine
  signal transduction (inter-cell communication)
• Previous data says Jak2 highest in unfertilized
  oocytes then reduces after 4-cell stage
• Present data shows that the level of this gene
  slightly increases at 1- and 2-cell stages and
  then reduces after 4-cell stage
• LDH (Lactate dehydrogenase) enzyme that converts
  lactate to pyruvate metabolism
• Reported to be highest in zygote (1-cell) stage
  and undetectable after 8-cell stage
• Consistent with in-virto but in-vivo data does
  not peak at zygote
• Overall pattern is same in in-vivo and in-vitro

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Comparisons of gene expression patterns between
cultured embryos and in-vivo embryos

• Hexokinase is a catalyst for glycolysis
• It increases during later stages of human or
  mouse embryo development
• Previous study shows increased hexokinase level
  in blastocysts
• Current data shows a peak at 8-cell stage and
  decrease after compaction
• Alpha 6 Integrin adhesion molecules (play a
  role in attachment of cell to extra-cellular
  matrix)
• Strong presence in the zygote stage
• The expression pattern of this gene for in-vivo
  and in-vitro embryos are comparable

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Comparisons of gene expression patterns between
cultured embryos and in-vivo embryos

• Cadherin-11 mediates cell adhesion
• Both data differ slightly from previous data
  where gene was detected only in blastocyst.
• Peaks at the blastocyst stage
• In-vivo and In-vitro gene expressions are a bit
  different for this data
• DNAMT1 (DNA methyltransferase 1) is an enzyme
  involved in DNA methylation
• Not expressed in embryo after implantation but
  expressed later in somatic proliferating cells
• In this study the in-vitro data shows unique
  expression pattern
• In-vivo data has no such peaks

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Conclusions

• These microarrary-based expression studies gave
  us a big picture of early embryo development
• These studies provide an opportunity to discover
  important genes involved in critical events that
  determine future organism structure
• In most stages of development there was minimal
  difference in expression patterns between in
  vitro and in vivo embryos