PostFertilization Development

1
Post-Fertilization Development Cleavage
Multicellular Organization
2
Cell Cycle
3
Cell cycle checkpoints controlled
by cyclin-dependent kinases (CDK) CDK
activates a transcription factor
by removing inhibition transcription of genes
necessary for next step in cycle
4
(No Transcript)
5
Go arrested stage forming cell pool which can
be activated later
Development of specialized cell types
6
Cleavage
7
- Rate of Cleavage in Rana pipiens -
Log cells/embryo
0 40 80 120 160
Hours Post-Fertilization
8
Cleavage
9
- Rate of Cleavage in Rana pipiens -
Log cells/embryo
0 40 80 120 160
Hours Post-Fertilization
10
Cleavage is an Epigenetic Process

• cell-to-cell interactions gap junctions

• positional information

11
Protein synthesis
12
Nuclear vs. Cytoplasmic Control
13
Hans Spemann - 1938
Different stages of development
Fig. 10-16, pg. 303
14
Genetic marker
- Nuclear transplantation experiment
- First series of cloning studies Gurdon 1968
15
Pre-existing label
Newly incorporated label

• Nucleus from neurula stage
• embryo transplanted into an
• egg

• Transplanted nucleus halts
• RNA synthesis

• When blastocyst stage is
• reached nucleus starts to
• synthesize large RNA
• molecules

• At neurula stage the nucleus
• is again synthesizing rRNA

16
Conclusions
17
Fig. 7.33, pg. 211
Cytoplasmic segregation Spemann 1938
Fig. 10.17, pg. 304
18
Organizer and Mesoderm Induction
Low Xnr have high BMP-4 Xwnt-8
High Xnrs activate other genes
creating Organizer region
Xnr protein concentration
Synergistic
Fig. 10.26, pg. 312
19
Sea Urchin embryo
Morphogenetic Gradient Theory
Fig. 8.6, pg. 217
20
Fig. 8.8, pg. 219
21
Conclusion
22
Sea Urchin Fate Map
Conklin 1905
Fig. 3.8, pg. 55
23
Lineage Fate Chart of Tunicate Embryo

• Hypothesis morphogen
• isolated in some cells and
• not others during division
• activates specific genes
• later in development

One half of the embryo represented
Fig. 8.8, pg. 219
24
Biochemical evidence for the existence
of cytoplasmic morphogens

• - Expose cells at
• different time periods
• to cytochalasin B
• Culture embryos for
• the time period required
• to form muscle cells
• 64-cell stage
• - stain for Achase

1
4
2
16
32
8
64
Tunicate embryos test for the presence of
Acetylcholinesterase
25
Lineage Fate Chart of Tunicate Embryo

• Hypothesis morphogen
• isolated in some cells and
• not others during division
• activates specific genes
• later in development

Cell-specific marker Acetylcholinesterase
Achase
One half of the embryo represented
Fig. 3.8, pg. 219
26
Cell cytoskeleton
27
Hypothesis - Differentiation due to
differential gene expression as development
proceeds
- Differentiation ----
Dedifferentiation ----
Redifferentiation
28
Metaplasia
Salamander eye
29
Nucleic Acid Hybridization and Gene Cloning
30
Labeled nucleotides

• complementary DNA can now be
• used as a molecular probe because it
• will hybridize with the mRNA sequence
• it was derived from (it is essentially the gene
  
• sequence for this product)

31
Molecular Cloning of Gene
Plasmid circular, double-stranded bacterial DNA
serves as the cloning
vector - incubate with E. coli to
replicate - isolate plasmids and cut out
cDNA
32
Polymerase Chain Reaction
Fig. 4.12, pg. 88
33
cDNA Subtraction Cloning
Stage-specific library
34
cDNA Library

• A compilation of all of the cDNAs that reflect
• the mRNAs present for a particular stage in
• development

35
Microarray Technique
Blastula-stage cell mRNA pool
Neurula-stage cell mRNA pool
mRNA in probe abundant in Cell type 1 mRNA in
probe abundant in cell type 2 mRNA in probe
abundant in both cell types
cDNA of mRNA from cell stage (or cell type) of
interest printed on to glass slide
probes e.g. Gastrula-stage cell
Fig. 4.13, pg. 90
36
In situ Hybridization using cDNA Probe
Fig. 4.16, pg. 92
37
Pax6 gene expression in the chicken
Pax6 mRNA highlighted in purple
38
Northern Blot
- mRNA from specific stages isolated by
elec- trophoresis and baked on to nitrocellulose
paper - incubated with DNA from
gastrula- specific cDNA clone
Developmental dot blot transcriptional activity
of 17 Xenopus genes
39
Northern Blot
mRNA Separation by Gel Electrophoresis
mRNA Blot on Membrane
Each lane in the gel contains the mRNA from a
specific cell of interest
Autoradiography
40
Southern Blot
Single stranded DNA separated by gel
electrophoresis
MW
41
Western Blot
42
Gene Knockout Technique
Fig. 4.19, pg. 95
43
Enhancer region
Promoter region regulates gene expression by
controlling binding of RNA polymerase Enhancer
region regulates activity of promoter
44
enhancer
45
Transgenic animals
Fig. 4.18, pg. 94
46
Gene Insertion into a Cell
47
Switch Genes
MyoD1 protein
Myoblast-specific cDNA clone MyoD1
protein serves as a regulatory protein which
binds to DNA and controls expression of
muscle-specific proteins
48
Fast twitch and slow twitch muscle fibers
Fast twitch fibers are more easily fatigued
sprinters develop these fibers for
explosive, short activity Slow twitch fibers
very resistant to fatigue (split ATP slowly)
endurance runners develop these fibers
Slow twitch fibers can be converted in to
fast twitch fibers by turning on the fat switch
gene Have taken the promoter region (the master
switch region) and spliced it in to mouse cells
which acts to keep this gene on continuously
49
Transgenic Animals
50
(No Transcript)
51
Transgenic Mice
Transgenic Cows
52
(No Transcript)
53
Regulation of Gene Expression
54
mRNA binding to ribosome
(heterogeneous nuclear RNA - hnRNA)
Stabilizes transcript, helps transport to
cytoplasm, regulates translation cleaved
Fig. 5.3, pg. 105
55
snRNP small nuclear ribonuclear proteins
56
Troponin and a-Tropomyosin
Splicing
18 exons encoding 259 amino acids / spliced to
produce 64 different mRNAs
13 exons variably spliced in different cell types
brain, striated muscle(2), smooth muscle,
myoblast, fibroblast, liver
Striated muscle
Smooth muscle
57
Drosophila Dscam gene

• predominantly involved in axon development and
  directing axons to
• their target cells

24 exons but many alternative adjacent DNA
sequences for 4 of the exons
38,016 different proteins possible
Dscam gene homologous to human gene on chromosome
21 expressed in nervous system -------------
aberrations lead to neurological defects seen in
Down syndrome
58
3-Day old chick
In situ hybridization with labeled
RNA complimentary to FGF8 mRNA
- mRNA for FGF8 exists in 7 different isoforms,
each product of which binds to a different FGF
receptor
59
Cell-specific Spliceosomes
1
2
3
4
5
6
Thyroid C cells
Brain
1 2 3 4
1 2 3 5 6
60
Sex Determination in Drosophila
Feminizing switch gene Sex-lethal (Sxl)
Fig. 17.16, pg. 546
61
(No Transcript)
62
Fig. 17.15, pg. 545
63

• Regulation of mRNA degredation
• Ribonucleases degrade RNA molecules
• - some proteins bind to mRNA to
• enhance RNAase activity and others
• bind and protect against RNAase
• Stabilization of Transcript for
• prolonged Translation

64
Half-life 28.5 hrs.
Casein degredation
Half-life 1.1 hrs.

• Incubate mammary tissue with 3H-Uridine to label
  casein mRNA
• Pulse for 3 hrs. / chase with cold Uridine
• Extract mRNA and analyze for casein mRNA with
  cDNA

Fig. 5.32, pg. 130
65
(hnRNA)
Fig. 5.3, pg. 105
66
?-LPH
?-end
N-terminal
ACTH
Anterior Lobe
Very Slow
?-LPH
N-terminal ACTH
?-LPH ?-LPH
Signal Sequence
Signal Sequence N-terminal ACTH
?-LPH
Signal Sequence
?-LPH
N-terminal ACTH
?-LPH ?-LPH
Very Fast
Intermediate Lobe
N-terminal
ACTH
?-LPH
?-end
?-MSH
CLIP
67
Proinsulin
68
(No Transcript)
69
Sex determination is hormone-dependent
Fig. 17.21, pg. 550
70
Sox9 gene expression Temperature
sensitive
Induction of Ovarian Development
71
Regulatory Mechanisms/Techniques to Date

• Gene amplification (vs. gene replication)
• Hormones (changing concentrations /-
  feedback)
• Hormone receptors (presence/absence/concentrati
  ons)
• Up/down regulation of gene expression
• Gap junctions (cAMP)
• Allosteric regulation of enzyme activity
• Paracrine control (hormones/gap junctions)
• Stabilization of mRNAs
• Turnover rates (half life)
• Presynthesis of mRNA (transcription) and
  storage
• for later translation (unequal
  positional storage)

• Antibodies 1st Ab, 2nd Ab, complexed with
• enzymes, fluorescent dyes Monoclonal
• antibodies
• Fluorescent molecules as substrates (Ca,
• PKC inhibitor)
• Selective Inhibitors Cycloheximide,
  Actinomycin D,
• Cytochalasin B

• Purify protein and synthesize mRNA and then cDNA
  using reverse
• transcriptase
• Use of plasmids for synthesizing large
  quantities of DNA sequence
• PCR method for synthesizing large quantities of
  DNA sequence
• Labeled cDNA probes subtraction cloning
• Microarray analyses
• Northern blots, southern blots, western blots
• Gene Knockout technique
• Transgenic animals
• Transcription regulation/Post-transcriptional
  regulation/Translational regulation/
• Post-translational regulation