Genome-Wide Studies of Early Embryo Development by Cd3PvA


									 Genome-Wide Studies of
Early Embryo Development

 Rong Kong, Nell Keith, Rohith Reddy
             Genome-Wide Studies of
              Embryo Development
   Questions:
       Which genes are important in early embryo
       What are the expression patterns of these genes
        over time?
       Does in-vitro culture affect embryo development?

   Methods:
       Genome-wide studied on preimplantation mouse
       In vivo and in vitro
               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
    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
              Microarray Study
   Expression-based, not phenotype-based
   High throughput compared with other methods
   Overall picture
   Quantitative results

   Difficult to obtain sufficient RNA in early embryo
   Require linear amplification

Microarray-based gene expression studies had
been done in C. elegans and Drosophila.
Stages of Early Development
Germinal Vesicle (GV) and metaphase II oocyte

In animals, fertilization is the
process that a sperm fuses with
an ovum, which eventually leads
to the development of an embryo.
    Zygotic Genome Activation (ZGA)
 The onset of new transcription
 Degradation of maternal transcripts
 ZGA happens during the transition from oocyte to

                               Examples of maternal
                                   • zp1-3 : zona pellucida
                                   • tPA: oocyte maturation
                                      in meiosis II
    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      12~32 cells
significant growth, producing a
cluster of cells the same size as
the original zygote.
 Compaction                      Adhesion    molecules
 A process that individual
 cells begin to adhere tightly        ZO-1, Cx43
                                      integrin, Cadherin
A strucuture consisting of an inner cell mass (or embryoblast) and an
outer cell mass (or trophoblast).

                                       ICM => embryonic stem cells
    Mid-preimplantation gene activation (MGA)

 Another wave of gene activation
 Companied by the dramatic morphological
 From compaction to morulae and blastocyst
A process in which blastocyst adheres to and enters into the uterine wall.
Some Signal Pathways

Wnt pathway   Notch pathway
 Developmental   Biology (7th Edition).
  Gilbert SF.
 Principles of Development. Wolpert L.
 Molecular Biology of the Cell. 4th Edition.
  Alberts B, et al.
 Wikipedia
“A Genome-Wide Study of
  Gene Activity Reveals
 Developmental Signaling
     Pathways in the
  Preimplantation Mouse
 Wang et. al., Developmental Cell, 2004
   Examine variation in overall gene expression levels
    during 12 stages of embryogenesis

   In vivo study

   Relate expression profile of specific genes to biological

   Explore conserved regulators of cell fate in embryo
       Wnt pathway
       Notch pathway
  Gene Expression Consistency in
   Each Individual Embryo Stage

• Embryos   staged based on morphological criteria

• Transcription profiles examined with Affymetrix Mouse

• Pearson correlation examined to assess similarity between
Gene Expression Variation in
Consecutive Embryo Stages
                 Pearson correlation
                  between consecutive
                  embryo development

                 Steeper curve
                  indicates greater
                  change in transcript
3-Fold Change in Gene Expression
     Between Embryo Stages
   Significant
    degradation of
    maternal mRNA
    during meiotic

   Changes in gene
    expression less
    pronounced after
Relative Levels of Transcription

   Determined percentage of genes receiving “present calls” (P call %)

   P call % decrease during meiotic maturation due to maternal mRNA

   After compaction and embryo layer development changes in P call %
    are difficult to detect
Hierarchical Clustering
               12 stages divided into
                two groups

               Change in gene
                expression reflects
                development of
     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
     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?
     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.
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

                         Relative expression levels
                            Blue = low level
                            Red = high level

                         Colored coded genes involved
                          in embryo development
                             Blue = resume cell cycle
                             Red = establish cell polarity
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
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
        Wnt and Notch Pathways
                                   Stages of activity:
                                       oocyte to embryo
                                       preceding implantation

   Work in conjunction
       Wnt promotes division
       Notch promotes
    The Wnt Signaling Pathway Is Active at the
                Blastocyst Stage

                                                    Inner Cell Mass


   RNA encoding β-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
  Gene expression in
preimplantation embryos
   from In-vitro study
 Paper by Shunping Wang et al.,
  Reproductive Biomedicine Online, 2005
   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
   Conclusion
Hierarchical clustering of gene expression at
       various developmental stages

   Results from Hierarchical clustering
   Clustering based on similarity of gene patterns
   Distance: Pearson’s correlation coefficient, centroid linkage method used
   Note the closeness and some intermixing of the 8-cell and compacting embryos, which indicates similar
    expression profiles
    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
    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
   Note the similar trends of up-regulated genes for both in vivo and in vitro., except the
    stage between 2- and 4-cell stage.
         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
   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
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
                                Low till 2-cell stage and increases
                                 till compacting stage
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
Comparisons of gene expression patterns between
    cultured embryos and in-vivo embryos
                           Hexokinase is a catalyst for
                                It increases during later stages of
                                 human or mouse embryo
                                Previous study shows increased
                                 hexokinase level in blastocysts
                                Current data shows a peak at 8-
                                 cell stage and decrease after

                           Alpha 6 Integrin – adhesion
                            molecules (play a role in
                            attachment of cell to extra-cellular
                                Strong presence in the zygote
                                The expression pattern of this
                                 gene for in-vivo and in-vitro
                                 embryos are comparable
Comparisons of gene expression patterns between
    cultured embryos and in-vivo embryos
                           Cadherin-11 mediates cell
                                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
                                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
   These microarrary-based expression studies
    gave us a big picture of early embryo

   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

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