Genome-Wide Studies of Early Embryo Development by Cd3PvA

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									 Genome-Wide Studies of
Early Embryo Development

 Rong Kong, Nell Keith, Rohith Reddy
            BIOE598SZ
            10/11/2006
             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
               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
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.
Stages of Early Development
               Oogenesis
Germinal Vesicle (GV) and metaphase II oocyte
                      Fertilization
                            Zygote




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
  embryo

                               Examples of maternal
                                  transcripts
                                   • zp1-3 : zona pellucida
                                      glycoproteins
                                   • tPA: oocyte maturation
                                      in meiosis II
                           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      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
                   Blastocyst
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
  changes
 From compaction to morulae and blastocyst
  stages
                     Implantation
A process in which blastocyst adheres to and enters into the uterine wall.
Some Signal Pathways




Wnt pathway   Notch pathway
               References
 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
        Embryo”
 Wang et. al., Developmental Cell, 2004
                        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
  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
Gene Expression Variation in
Consecutive Embryo Stages
                 Pearson correlation
                  between consecutive
                  embryo development
                  stages

                 Steeper curve
                  indicates greater
                  change in transcript
                  composition
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
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
Hierarchical Clustering
               12 stages divided into
                two groups

               Change in gene
                expression reflects
                morphological
                development of
                embryo
     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
                          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
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
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
        differentiation
    The Wnt Signaling Pathway Is Active at the
                Blastocyst Stage


                                                    A-C:
                                                    Inner Cell Mass



                                                    D-F:
                                                    Trophectoderm



   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
                   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
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
    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.
         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
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
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
                            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
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
                 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

								
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