PostFertilization Development

1
Post-Fertilization Development Cleavage
Multicellular Organization
2
Gap 1 enzymes and nucleotides required for DNA
replication synthesized
M
Mitosis Two 2n cells produced
Synthesis Stage DNA replication
Gap 2 proteins required for mitosis synthesized
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
Alteration of checkpoint inhibitors loss of
cycle regulation CANCER
4
Duration of Cell Cycle for different cell
types?
Pulse/Chase Approach 3H-thymidine pulse _at_ T
0 Chase with cold thymidine
5
3H-thymidine pulse _at_ T 0 Chase with
cold thymidine
G2 Time from T 0 to appearance of
labeled mitotic cells
S Time between 1st appearance of labeled
mitotic cells and their disappearance
M Duration determined from direct visual
measurement
PML of mitoses which are labeled
G1 Solve from equation
Total duration of cell cycle G1 S G2 M
6
Go arrested stage forming cell pool which can
be activated later
Development of specialized cell types
7
Cleavage
8
- Rate of Cleavage in Rana pipiens -
Log cells/embryo
0 40 80 120 160
Hours Post-Fertilization
9
Cleavage
10
- Rate of Cleavage in Rana pipiens -
Growth phase as differentiation begins at
blastulation
Log cells/embryo
No growth phase following mitosis
0 40 80 120 160
Hours Post-Fertilization
11
Cleavage is an Epigenetic Process

• cell-to-cell interactions gap junctions

• positional information

12
Enzymes for cleavage Actin for microfilaments Tubu
lin for microtubules
Protein synthesis
13
Nuclear vs. Cytoplasmic Control
14
Hans Spemann - 1938
Different stages of development
Conclusion ?
Nuclear Equivalence
Fig. 10-16, pg. 303
15
Genetic marker
- Nuclear transplantation experiment
- First series of cloning studies Gurdon 1968
16
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

17
Conclusions
- Nucleus of differentiated cell retains all
genetic information necessary for development
Cell cytoplasm regulates nuclear
expression
Hypothesis Cellular differentiation and
specialization may be the result of
cytoplasmic segregation during cleavage
18
Constriction of fertilized egg/16-cell nuclei
incorporated
Gray crescent
Fig. 7.33, pg. 211
Cytoplasmic segregation Spemann 1938
Fig. 10.17, pg. 304
19
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
20
Sea Urchin embryo
Morphogenetic Gradient Theory
Fig. 8.6, pg. 217
21
Fig. 8.09, pg. 219
22
Conclusion
Cell-to-cell communication along a gradient
through the synthesis and secretion of morphogens
Differentiation vs. Determination
23
Sea Urchin Fate Map

• Neural tissue
• does not form
• unless both Animal
• and Vegetal pole
• cells interact
• Occurs at 64-cell
• stage

Conklin 1905
Fig. 3.8, pg. 55
24
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. 8.8, pg. 219
25
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
26
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
Expose 32-cell stage embryo to Actinomycin D
----------- No Achase activity at 64-cell
stage!
One half of the embryo represented
Fig. 8.8, pg. 219
27
Cell cytoskeleton
28
Hypothesis - Differentiation due to
differential gene expression as development
proceeds
- Differentiation ----
Dedifferentiation ----
Redifferentiation
29
Fig. 6.4b, pg. 146
Competence / Bias / Determination
Fig. 6.5a, pg. 147
30
Metaplasia
1) Dorsal iris cells produce enormous
amounts of ribosomes 2) Cell DNA
replication occurs 3) Dedifferentiate (lose
melanosomes) 4) Redifferentiation crystallin
protein synthesis
Day 5
Day 7
Day 9
Day 16
Day 18
Day 30
Salamander eye
31
Nucleic Acid Hybridization and Gene Cloning
32
Labeled nucleotides
RNA Polymerase
Transcription
33
Labeled nucleotides

• complementary DNA (cDNA) 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)

34
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
35
Polymerase Chain Reaction - PCR
Taq polymerase isolated from unique
bacteria (Thermus aquaticus) living in thermal
pools in Yellowstone NP Thermostabile to near
boiling
Fig. 4.12, pg. 88
36
cDNA Subtraction Cloning
Stage-specific library Gastrula-specific
library
Or use PCR approach
37
cDNA Library

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

- Evolutionary studies Is the morphogen
for ectoderm development in the mouse the
same as found in the chicken ? - Developmental
expression In what cell is a particular
gene expressed and when ?
38
In situ Hybridization using cDNA Probe
Fig. 4.16, pg. 92
39
Pax6 gene expression in the chicken
Pax6 mRNA highlighted in purple
40
Northern Blot - mRNA
mRNA
mRNA
mRNA
Cell with stage-specific mRNA
mRNA
mRNA
mRNA Separation by Gel Electrophoresis
mRNA Blot on Membrane
mRNA

Each lane in the gel contains the mRNA from a
specific cell of interest
_
Add specific labeled
cDNA probe
Autoradiography
41
Northern Blot
- mRNA from specific stages isolated and baked
on to nitrocellulose paper - incubated with
cDNA from gastrula- specific cDNA clone
Developmental dot blot transcriptional activity
of 17 Xenopus genes
42
Southern Blot - DNA
Single stranded DNA separated by gel
electrophoresis
MW
Add specific labeled
cDNA probe
43
Microarray Technique
Monitoring timing of transcrition for thousands
of genes simultaneously
Blastula-stage cell mRNA pool
Neurula-stage cell mRNA pool
Compare resultant fluorescent intensities
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 each individual mRNA from a
particular cell stage (or cell type) of interest
printed on to glass slide probes e.g.
Gastrula-stage cell
Fig. 4.13, pg. 90
44
Western Blot - proteins -
45
DNA repair mechanism incorporates this copy
sometimes
Gene Knockout Technique
1 isolate gene of interest (e.g., BMP7) 2
insert neomycin resistance gene into region
that encodes DNA- binding site of BMP7
(renders this gene product inactive) 3
insert this gene in to stem cells, culture
cells and isolate neomycin resistant ones
(have incorporated gene)
Wild type cells from host Heterozygyous Bmp7
cells from donor
Fig. 4.19, pg. 95
46
Cloned gene in Retroviral vector
Transgenic animals
Fig. 4.18, pg. 94
47
Gene Insertion into a Cell
Non-viral vector

• Transfection gene
• with regulatory
• regions cultured
• with cells -------
• - taken in to cells
• by endocytosis

Fig. 21.24, pg. 682
48

• Retroviral vector gene
• inserted into a retrovirus
• (RNA genome-containing
• virus) - retrovirus make
• DNA copy then replicate
• becoming double stranded
• and is incorporated into
• the cells chromosome
• (100 efficient)

Fig. 21.24, pg. 682
49
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
50
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 fast
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
51
Transgenic Animals
Flounder switch gene for antifreeze protein
attached to GH (active in cold water environment
year round) - grows twice as fast / disease
resistant / more prolific
Because of concerns about potential hybridization
with native salmon only triploid females will be
produced
52

• Created fluorescent zebra fish by inserting
  fluorescence gene
• from jellyfish used to track environmental
  pollution
• Are adding a "switch" that will cause the always
  fluorescing
• GloFish zebra fish to selectively fluoresce in
  the presence
• of environmental toxins. A non-fluorescing fish
  will signal that
• the water is safe, while a fluorescing fish will
  signal trouble.

Insert inducible gene promoters as control
switches estrogen- inducible promoter and a
stress-responsive promoter.
53
Ultraviolet light
Regular light
54
Transgenic chickens have been produced by
inserting human genes containing regulatory
elements ensuring proteins would only be made in
cells secreting egg-white proteins. Retroviral
vector used to insert genes into male chick
embryos bred resulting roosters with hens.
Eggs layed with high concentrations of target
proteins antibody to melanoma, interferon
to treat multiple sclerosis, interferon
used to treat Hepatitis C and some leukaemias
Inheritable.
55
Transgenic pigs produced that express a protein
that converts omega-6 fatty acids to omega-3
fatty acids reduces coronary heart disease and
lowers triglyceride levels.
56
Transgenic Mice Rat GH gene inserted
Transgenic Cows Human gene for blood platelets
inserted into mammary gland cells Increased
milk production and designer milk lactose- free
57
Spider genes for silk web proteins inserted into
goats linked to milk-producing
genes Bullet-proof clothing to tendons and
ligaments in humans
Xenotransplantation pig organs very similar to
humans pig cell-surface protein causes donor
rejection Therefore are removing pig protein
and replacing it with a human protein.
58
9 February 2009 10
A transgenic approach to speciation
heterologous expression of master genes coding
for species attributes provides a unique
approach to conservation through selective
species formation. Darwin, C. R., G. J.
Mendel, Sir M. P. Jagger, and
B. Bird (2009) 312774-781.
Brave New World From Ants to Monkeydogs
59
Transgenic Golden Retriever x
Sparrow
Darwin et al. Science (2008)
60
Transgenic Crocodile x Parrot
Darwin et al. Science (2008)
61
Transgenic Retriever x
Macque
Darwin et al. Science (2008)
62
Regulation of Gene Expression

• Transcriptional regulation

• Post-transcriptional modification

• Translational regulation

• Post-translational modification

63
Primary Gene Components
Promoter region
Enhancer region
Informational portion of gene
RNA polymerase
Promoter region regulates gene expression by
controlling binding of RNA polymerase Enhancer
region regulates activity of promoter
Fig. 5.2, pg. 104
64
enhancer
Enhancer elements
65
Fig. 5.2, Pg. 104
66
heterogenous nuclear RNA (hnRNA)
Fig. 5.2, Pg. 104
67
Spliceosomes
(heterogeneous nuclear RNA - hnRNA)
Posttranscriptional modification
mRNA binding to ribosome
Stabilizes transcript, helps transport to
cytoplasm, regulates translation cleaved
1 hnRNA gt 100 mRNAs
Fig. 5.3, pg. 105
68
snRNP small nuclear ribonuclear proteins
69
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
70
Drosophila Dscam gene

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

Proteomics
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
71
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
72
Cell-specific Spliceosomes
1
2
3
4
5
6
Thyroid C cells
Brain
1 2 3 4
1 2 3 5 6
Calcitonin mRNA for Calcitonin - lowers Ca
levels
Calcitonin-gene related peptide (CGRP) -
neuropeptide regulating gastric function
73
Sex Determination in Drosophila
Females are XX Males are XY Y gene expression
is NOT involved in sex determination Sex
determination revolves around female sex
determinants from X gene expression vs. male
determinants from autosomal (A) gene expression
Females 2X2A Males 1X2A
74
Sex Determination in Drosophila
Feminizing switch gene Sex-lethal (Sxl)
expressed within 2 hr. post fertilization
Feminizing switch gene Sex-lethal (Sxl)
inactive during early development
Fig. 17.16, pg. 546
75
(No Transcript)
76
msl gene regulates transcription from X
chromosome dosage compensation
Male X hypertranscription (dosage compensation)
does not start until after the sisterless gene
activity to measure the XA ratio is over.
Fig. 17.15, pg. 545
77
Dosage compensation Structure of X chromosome in
male is altered by acetylation of nucleosomes so
that promoter sites of genes become more exposed
and RNA polymerases can bind with higher
efficiency
78

• 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

79
Half-life 28.5 hrs.
Half-life 1.1 hrs.
Casein mRNA degredation

• 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

Prolactin thought to increase polyadenylation of
casein mRNA which decreases rate of degredation
Fig. 5.32, pg. 130
80
(hnRNA)
Spliceosomes
Fig. 5.3, pg. 105
81
beta-LPH
beta-end(31)
N-terminal
ACTH
Very Slow
Anterior Lobe
beta-LPH
N-terminal ACTH
?-LPH(58/90) beta-LPH
Signal Sequence
Signal Sequence N-terminal ACTH
beta-LPH(90)
(39 aa)
Signal Sequence
beta-LPH
N-terminal ACTH
?-LPH beta-LPH
Very Fast
N-terminal
ACTH
Intermediate Lobe
beta-LPH
beta-end
alpha-MSH(13)
CLIP(21)
82
84 a.a. peptide spliced to 51 a.a. functional
hormone
Proinsulin Insulin
83

• Linear Sequence
• of Amino Acids

• alpha Helix/
• beta-Pleated sheet

• Globular Proteins
• Hydrogen bonds/
• Ionic bonds/
• Disulfide bridges/

• Oligomeric Proteins
• 2 or more proteins

Regulation of Turnover Rate
84
Sex determination

• Genetic dependent (GSD)
• Temperature dependent (TSD)
• Both can be influenced by other
• environmental or behavioral mechanisms

85
Sex determination in most turtles and all
crocodilians is hormone-dependent
Enzymatic control of hormone synthesis
regulated by temperature- sensitive expression
of Sox9 gene
Red-eared slider turtle
Alligator snapping turtle
Nest under sand
Fig. 17.21, pg. 550
86
Enzymatic control of hormone synthesis regulated
by temperature- sensitive expression of Sox9 gene
Sex determination in most turtles and all
crocodilians is hormone-dependent
Red-eared slider turtle
Alligator snapping turtle
Nest under sand Temperature gradient
Fig. 17.21, pg. 550
87
_at_ Male-promoting temperatures --- Up regulation
of Sox9 expression which acts as an
inhibitory transcription factor for aromatase
gene expression _at_ Female-promoting
temperatures--- Down regulation of Sox9 gene
expression which removes inhibition and allows
subsequent aromatase activity
(Luteinizing hormone)
Sox9 gene expression Temperature
sensitive
Induction of Ovarian Development
88
Climate change and temperature-dependent sex
determination in reptiles. F. J. Janzen, (1994).
Proc. Natl. Acad. Sci. 917487-7490.
Fig. 1. Annual cohort sex ratio ( male) of
hatchling painted turtles (Chrysemys picta) from
1988 to 1993 as a function of the mean ambient
temperature in July.
89
Ecology meets endocrinology environmental sex
determination in fishes. Godwin, Luckenbach, and
Borski. (2003). Evolution Development 540-49.
5050 sex ratio
Flounder sex ratios resulting from development at
different temperatures. Depicted are data from
barfin flounder (), japanese flounder (), and
southern flounder (?). References are in text.
Japanese flounder values are means calculated
from Yamamoto (1999).
Warm and cold currents in the area of Japan. The
range of the japanese flounder (Paralichthys
olivaceus) extends from northern Japan south to
Hong Kong. The range of the barfin flounder
(Verasper moseri) extends from the Kuril Islands
north of Japan south to approximately the point
where the Oyahsio and Kuroshio currents are shown
meeting.
90
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

Darwin et al. Science (2008)