Cellular Mechanisms
of Development
Chapter 19
Overview of Development
Development is the successive process of
systematic gene-directed changes
throughout an organism’s life cycle
-Can be divided into four subprocesses:
-Growth (cell division)
-Differentiation
-Pattern formation
-Morphogenesis
2
Cell Division
After fertilization, the diploid zygote undergoes
a period of rapid mitotic divisions
-In animals, this period is called cleavage
-Controlled by cyclins and cyclindependent kinases (Cdks)
During cleavage, the zygote is divided into
smaller & smaller cells called blastomeres
-Moreover, the G1 and G2 phases are
shortened or eliminated
3
Cell Division
4
Cell Division
Adult Cell Cycle
Cell Cycle of Early Frog Blastomere
Mitosis
Active
M
Cdk /G2
cyclin
C
Cdk /
cyclin
G1 Cdk /G1 Active
cyclin
C
M
G2
Active
Cdk /
S
Active cyclin
M
Cyclin
Synthesis
S
DNA Synthesis
a.
Mitosis
S
G2
M
C
interphase
Cyclin
Degradation
S
Inactive
S
Cdk
mitosis
cytokinesis
DNA Synthesis
b.
5
Cell Division
Caenorhabditis elegans
-One of the best developmental models
-Adult worm consists of 959 somatic cells
-Transparent, so cell division can be
followed
-Researchers have mapped out the lineage
of all cells derived from the fertilized egg
6
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Nematode Lineage Map
Egg
Egg and
sperm line
Nervous
system
Pharynx
Intestine
Cuticle-making cells Vulva
Gonad
a.
Adult Nematode
Gonad
Egg
Vulva
b.
Gonad
Intestine
Sperm
Cuticle
Nervous system
Pharynx
7
Cell Division
Blastomeres are nondifferentiated and can
give rise to any tissue
Stem cells are set aside and will continue to
divide while remaining undifferentiated
-Tissue-specific: can give rise to only one
tissue
-Pluripotent: can give rise to multiple
different cell types
-Totipotent: can give rise to any cell type 8
Cell Division
Cleave in mammals continues for 5-6 days
producing a ball of cells, the blastocyst
-Consists of:
-Outer layer = Forms the placenta
-Inner cell mass = Forms the embryo
-Source of embryonic stem cells
(ES cells)
9
Once sperm cell and egg cell have joined, cell
cleavage produces a blastocyst. The inner cell mass
of the blastocyst develops into the human embryo.
Embryonic stem-cell
culture
Inner cell
mass
Egg
Sperm
Blastocyst
Embryo
Embryonic stem cells (ES cells) are
isolated from the inner cell mass
10
Cell Division
A plant develops by building its body outward
-Creates new parts from stem cells
contained in structures called meristems
-Meristematic stem cells continually divide
-Produce cells that can differentiate into
the various plant tissues
-Leaves, roots, branches, and flowers
The plant cell cycle is also regulated by
cyclins and cyclin-dependent kinases
11
Cell Differentiation
A human body contains more than 210 major
types of differentiated cells
Cell determination commits a cell to a
particular developmental pathway
-Can only be “seen” by experiment
-Cells are moved to a different location in
the embryo
-If they develop according to their new
position, they are not determined 12
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Normal
Not Determined
(early development)
No donor
Donor
Tail cells are
transplanted
to head
Recipient
Before Overt
Differentiation
Determined
(later development)
Tail
Tail cells are
transplanted
to head
Head
Recipient
After Overt
Differentiation
Tail cells develop
into head cells in head
Tail cells develop
into tail cells in head
13
Cell Differentiation
Cells initiate developmental changes by using
transcriptional factors to change patterns of
gene expression
Cells become committed to follow a particular
developmental pathway in one of two ways:
1) via differential inheritance of cytoplasmic
determinants
2) via cell-cell interactions
14
Cell Differentiation
Cytoplasmic determinants
-Tunicates are marine invertebrates
-Tadpoles have tails, which are lost during
metamorphosis into the adult
-Egg contains yellow pigment granules
-Become asymmetrically localized
following fertilization
-Cells that inherit them form muscles
15
MEIOSIS
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Sperm
(haploid) n
Egg
(haploid) n
Adult tunicate
(diploid) 2n
n
Pigment
granules
2n
Embryo
(diploid) 2n
Larva
(diploid) 2n
a.
16
Cell Differentiation
Cytoplasmic determinants
-Female parent provides
egg with macho-1 mRNA
-Encodes a transcription
factor that can activate
expression of musclespecific genes
17
Cell Differentiation
Induction is the change in the fate of a cell
due to interaction with an adjacent cell
If cells of a frog embryo are separated:
-One pole (“animal pole”) forms ectoderm
-Other pole (“vegetal pole”) forms endoderm
-No mesoderm is formed
If the two pole cells are placed side-by-side,
some animal-pole cells form the mesoderm
18
Cell Differentiation
Another example of induction is the formation
of notochord and mesenchyme in tunicates
-Arise from mesodermal cells that form at
the vegetal margin of 32-cell stage embryo
-Cells receive a chemical signal from
underlying endodermal cells
-Anterior cells differentiate into notochord
-Posterior cells differentiate into
mesenchyme
19
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Sagittal section Longitudinal
section
1 2
1
Anterior
Posterior
Dorsal nerve cord (NC)
Anterior
Ventral endoderm (En) Notochord (Not)
Longitudinal 2
section
Mesenchymal cells (Mes)
Posterior
Tail muscle cells (Mus)
a.
FGF signaling
Anterior
Posterior
32-Cell Stage
64-Cell Stage
20
Cell Differentiation
The chemical signal is a fibroblast growth
factor (FGF) molecule
-The FGF receptor is a tyrosine kinase that
activates a MAP kinase cascade
-Produces a transcription factor that
triggers differentiation
Thus, the combination of macho-1 and FGF
signaling leads to four different cell types
21
Cell Differentiation
FGF
FGF Receptor
Cell membrane
First Step
Second Step
Cell Types
Yes
FGF Signal received?
Yes
No
No
FGF Signal received?
Yes
No
Macho-1 inherited?
Ras/MAPK
Pathway
Mesenchyme
Muscle
Notochord
T-Ets
Macho-1
P
Nerve cord
a.
Suppression of muscle
genes and activation
of mesenchyme genes
Mesenchyme
Precursor Cells
b.
22
Cell Differentiation
No
FGF
FGF Receptor
FGF Receptor
Cell membrane
Ras/MAPK
Pathway
T-Ets
No
FGF
FGF
Cell membrane
Ras/MAPK
Pathway
T-Ets No Macho-1
Macho-1
FGF Receptor
Cell membrane
Ras/MAPK
Pathway
T-Ets
No Macho-1
P
Transcription of
muscle genes
Muscle
Precursor Cells
Transcription
of notochord genes
Notochord
Precursor Cells
Suppression of notochord
genes and activation
of nerve cord genes
Nerve cord
Precursor Cells
23
Cloning
Until very recently, biologists thought that
determination and cell differentiation were
irreversible in animals
Nuclear transplant experiments in mammals
were attempted without success
-Finally, in 1996 a breakthrough
Geneticists at the Roslin Institute in Scotland
performed the following procedure:
24
Cloning
1. Differentiated mammary cells were removed
from the udder of a six-year old sheep
2. Eggs obtained from a ewe were enucleated
3. Cells were synchronized to a resting state
4. The mammary and egg cells were combined
by somatic cell nuclear transfer (SCNT)
5. Successful embryos (29/277) were placed in
surrogate mother sheep
6. On July 5, 1996, Dolly was born
25
Preparation
Cell Fusion
Cell Division
Mammary cell is extracted and grown in nutrientdeficient solution that arrests the cell cycle.
Mammary cell is Electric shock fuses cell
inserted inside
membranes and triggers
Nucleus containing
covering
of
egg
cell.
cell division.
source
Egg cell is extracted.
Nucleus is removed from
egg cell with a micropipette.
Development Implantation
Embryo begins to
develop in vitro.
Embryo
Embryo is
implanted into
surrogate
mother.
Birth of Clone
Growth to Adulthood
After a five-month pregnancy, a
lamb genetically identical to the
sheep from which the mammary
cell was extracted is born.
26
Cloning
Dolly proved that determination in animals is
reversible
-Nucleus of a differentiated cell can be
reprogrammed to be totipotent
Reproductive cloning refers to the use of
SCNT to create an animal that is genetically
identical to another
-Scientists have cloned cats, rabbits, rats,
mice, goats and pigs
27
Cloning
Reproductive cloning has inherent problems
1. Low success rate
2. Age-associated diseases
Normal mammalian development requires
precise genomic imprinting
-The differential expression of genes based
on parental origin
Cloning fails because there is not enough time
to reprogram the genome properly
28
Cloning
In therapeutic cloning, stem cells are cloned
from a person’s own tissues and so the body
readily accepts them
Initial stages are the same as those of
reproductive cloning
-Embryo is broken apart and its embryonic
stem cells extracted
-Grown in culture and then used to
replace diseased or injured tissue
29
Cloning
The nucleus from a skin cell of a diabetic
patient is removed.
The skin cell
Cell cleavage
nucleus is inserted occurs as the
into the enucleated embryo begins to
human egg cell. develop in vitro.
The embryo
reaches the
blastocyst stage
Inner cell
mass
Diabetic
patient
The nucleus from a skin cell of a healthy
patient is removed.
ES
Early embryo cells
Blastocyst
Healthy
patient
30
Cloning
Therapeutic Cloning
Embryonic stem cells
(ES cells) are extracted
and grown in culture.
The stem cells are developed
into healthy pancreatic islet cells
needed by the patient.
The healthy tissue is
injected or transplanted
into the diabetic patient.
Diabetic
patient
Healthy pancreatic islet cells
Reproductive Cloning
The blastocyst is kept intact and
is implanted into the uterus of a
surrogate mother.
The resulting baby is
a clone of the
healthy patient.
31
Cloning
Human embryonic stem cells have enormous
promise for treating a wide range of diseases
-However, stem cell research has raised
profound ethical issues
Very few countries have permissive policy
towards human reproductive cloning
-However, many permit embryonic stem cell
research
32
Cloning
Early reports on a variety of adult stem cells
indicated that they may be pluripotent
-Since then these
results have been
challenged
33
Pattern Formation
In the early stages of pattern formation, two
perpendicular axes are established
-Anterior/posterior (A/P, head-to-tail) axis
-Dorsal/ventral (D/V, back-to-front) axis
Polarity refers to the acquisition of axial
differences in developing structures
Position information leads to changes in
gene activity, and thus cells adopt a fate
appropriate for their location
34
Drosophila Embryogenesis
Drosophila produces two body forms
-Larva – Tubular eating machine
-Adult – Flying sex machine axes are
established
Metamorphosis is the passage from one
body form to another
Embryogenesis is the formation of a larva
from a fertilized egg
35
Drosophila Embryogenesis
Before fertilization, specialized nurse cells
move maternal mRNAs into maturing oocyte
-These mRNA will initiate a cascade of gene
activations following fertilization
Embryonic nuclei do not begin to function until
approximately 10 nuclear divisions later
36
Drosophila Embryogenesis
After fertilization, 12 rounds of nuclear division
without cytokinesis produces a syncytial
blastoderm
-4000 nuclei in a single cytoplasm
Membranes grow between the nuclei forming
the cellular blastoderm
Within a day of fertilization, a segmented,
tubular body is formed
37
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Embryo
Hatching larva
Movement of
maternal mRNA
Follicle
cells
Nurse
cells
Posterior
Oocyte
Anterior
Three larval stages
Nucleus
Fertilized egg
a.
c.
Metamorphosis
Syncytial blastoderm
d.
Cellular blastoderm
Nuclei line up along
surface, and membranes
grow between them to
form a cellular blastoderm.
Thorax
Head
Abdomen
Segmented embryo prior to hatching
b.
38
e.
Drosophila Embryogenesis
Nüsslein-Volhard and Wieschaus elucidated
how the segmentation pattern is formed
-Earned the 1995 Nobel Prize
Two different genetic pathways control the
establishment of the A/P and D/V polarity
-Both involve gradients of morphogens
-Soluble signal molecules that can
specify different cell fates along an axis
39
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Establishing the Polarity of the Embryo
Fertilization of the
egg triggers the
production of bicoid
protein from maternal
RNA in the egg. The
bicoid protein
diffuses through the
egg, forming a
gradient. This
gradient determines
the polarity of the
embryo, with the
head and thorax
developing in the
zone of high
concentration (green
fluorescent dye in
antibodies that bind
bicoid protein allows
visualization of the
gradient).
Bicoid
About 21/2 hours
after fertilization,
bicoid protein turns
on a series of brief
signals from socalled gap genes.
The gap proteins act
to divide the embryo
into large blocks. In
this photo,
fluorescent dyes in
antibodies that bind
to the gap proteins
Krüppel (orange)
and Hunchback
(green) make the
blocks visible; the
region of overlap is
yellow.
Laying Down the Fundamental Regions
About 0.5 hr later,
the gap genes
switch on the
“pair-rule” genes,
which are each
expressed in seven
stripes. This is
shown for the pairrule gene hairy .
Some pair-rule
genes are only
required for evennumbered
segments while
others are only
required for odd
numbered
segments.
Setting the Stage for Segmentation
Hairy
Krüppel
Hunchback
Forming the Segments
The final stage of
segmentation occurs
when a “segmentpolarity” gene called
engrailed divides
each of the seven
regions into halves,
producing 14 narrow
compartments. Each
compartment
corresponds to one
segment of the future
body. There are three
head segments (H,
bottom right), three
thoracic segments
(T, upper right), and
eight abdominal
segments (A, from
top right to bottom
left).
Engrailed
40
Establishment of the A/P axis
Nurse cells secrete maternally produced bicoid
and nanos mRNAs into the oocyte
-Differentially transported by microtubules to
opposite poles of the oocyte
-bicoid mRNA to the future anterior pole
-nanos mRNA to the future posterior pole
-After fertilization, translation will create
opposing gradients of Bicoid and Nanos
proteins
41
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Movement of bicoid mRNA moves
maternal mRNA toward anterior end
Follicle
cells
Nurse
cells
Anterior
Posterior
Microtubules nanos mRNA moves
toward posterior end
a.
Nucleus
Anterior
Posterior
bicoid
mRNA
b.
nanos
mRNA
42
Establishment of the A/P axis
Bicoid and Nanos control translation of two
other maternal mRNAs, hunchback and
caudal, that encode transcription factors
-Hunchback activates anterior structures
-Caudal activates posterior structures
The two mRNAs are not evenly distributed
-Bicoid inhibits caudal mRNA translation
-Nanos inhibits hunchback mRNA translation
43
Concentration
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nanos mRNA
hunchback mRNA
bicoid mRNA
caudal mRNA
Anterior
Posterior
a. Oocyte mRNAs
Anterior
bicoid mRNA
Bicoid protein
caudal mRNA
Caudal protein
Posterior
nanos mRNA
Nanos protein
hunchback mRNA
Hunchback protein
b. After fertilization
Concentration
Nanos protein
Hunchback protein
Bicoid protein
Caudal protein
Anterior
c. Early cleavage embryo proteins
Posterior
44
Establishment of the D/V axis
Maternally produced dorsal mRNA is placed
into the oocyte
-Not asymmetrically localized
Oocyte nucleus synthesizes gurken mRNA
-Accumulates in a crescent on the future
dorsal side of embryo
After fertilization, a series of steps results in
selected transport of Dorsal into ventral
nuclei, thus forming a D/V gradient
45
46
Production of Body Plan
The body plan is produced by sequential
activation of three classes of segmentation
genes
1. Gap genes
-Map out the coarsest subdivision along
the A/P axis
-All 9 genes encode transcription factors
that activate the next gene class
47
Production of Body Plan
2. Pair-rule genes
-Divide the embryo into seven zones
-The 8 or more genes encode
transcription factors that regulate each
other, and activate the next gene class
3. Segment polarity genes
-Finish defining the embryonic
segments
48
Production of Body Plan
Segment identity arises from the action of
homeotic genes
-Mutations in them lead to the appearance
of normal body parts in unusual places
-Ultrabithorax
mutants produce an
extra pair of wings
49
Production of Body Plan
Homeotic gene complexes
-The HOM complex genes of Drosophila
are grouped into two clusters
-Antennapedia complex, which governs
the anterior end of the fly
-Bithorax complex, which governs the
posterior end of the fly
-Interestingly, the order of genes mirrors the
order of the body parts they control
50
Production of Body Plan
Homeotic gene complexes
-All of these genes contain a conserved
180-base sequence, the homeobox
-Encodes a 60-amino acid DNA-binding
domain, the homeodomain
-Homeobox-containing genes are termed
Hox genes
-Vertebrates have 4 Hox gene clusters
51
Production of Body Plan
Drosophila HOM Chromosomes
Mouse Hox Chromosomes
Drosophila HOM genes
Antennapedia complex
Bithorax complex
Hox 2
lab pb Dfd Scr Antp
Ubxabd-Aabd-B
Hox 3
Head Thorax
a.
Hox 1
Hox 4
Abdomen
Fruit fly
embryo
Mouse
embryo
Fruit fly
Mouse
b.
52
Pattern Formation in Plants
The predominant homeotic gene family in
plants is the MADS-box genes
-Found in most eukaryotic organisms,
although in much higher numbers in plants
MADS-box genes encode transcriptional
regulators, which control various processes:
-Transition from vegetative to reproductive
growth, root development and floral organ
identity
53
Morphogenesis
Morphogenesis is the formation of ordered
form and structure
-Animals achieve it through changes in:
-Cell division
-Cell shape and size
-Cell death
-Cell migration
-Plants use these except for cell migration
54
Morphogenesis
Cell division
-The orientation of the mitotic spindle
determines the plane of cell division in
eukaryotic cells
-If spindle is centrally located, two
equal-sized daughter cells will result
-If spindle is off to one side, two
unequal daughter cells will result
55
Morphogenesis
Cell shape and size
-In animals, cell differentiation is
accomplished by profound changes in cell
size and shape
-Nerve cells develop long processes
called axons
-Skeletal muscles cells are large and
multinucleated
56
Morphogenesis
Cell death
-Necrosis is accidental cell death
-Apoptosis is programmed cell death
-Is required for normal development in
all animals
-“Death program” pathway consists of:
-Activator, inhibitor and apoptotic
protease
57
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Organism
Caenorhabditis elegans
Mammalian Cell
Inhibitor:
CED-9
Activator:
CED-4
Apaf1
Apoptotic
Protease:
CED-3
Caspase-8 or -9
Apoptosis
Apoptosis
Inhibition
Activation
a.
Inhibitor
Bcl-2
b.
58
Morphogenesis
Cell migration
-Cell movement involves both adhesion and
loss of adhesion between cells and substrate
-Cell-to-cell interactions are often mediated
through cadherins
-Cell-to-substrate interactions often involve
complexes between integrins and the
extracellular matrix (ECM)
59
Development of Seed Plants
Plant development occurs in five main stages:
1. Early embryonic cell division
-First division is off-center
-Smaller cell divides to form the embryo
-Larger cell divides to form suspensor
-Cells near it ultimately form the root
-Cells on the other end, form the shoot
60
Development of Seed Plants
2. Embryonic tissue formation
-Three basic tissues differentiate:
-Epidermal, ground and vascular
3. Seed formation
-1-2 cotyledons form
-Development is arrested
4. Seed germination
-Development resumes
-Roots extend down, and shoots up
61
Development of Seed Plants
5. Meristematic development and
morphogenesis
-Apical meristems at the root and shoot
tips generate a large numbers of cells
-Form leaves, flowers and all other
components of the mature plant
62
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Epidermal
cells
Ground
tissue cells
Vascular
tissue cells
Embryo
Embryo
Shoot apical meristem
Suspensor
Cotyledons
a. Early cell division
b. Tissue formation
Shoot apical
meristem
Seed wall
Cotyledons
Root apical
meristem
Root apical
c. Seed formation meristem
d. Germination
63
e. Meristematic development and morphogenesis
Environmental Effects
Both plant and animal development are
affected by environmental factors
-Germination of a dormant seed proceeds
only under favorable soil and day conditions
-Reptiles have a temperature-dependent
sex determination (TSD) mechanism
-The water flea Daphnia changes its shape
after encountering a predatory fly larva
64
Environmental Effects
65
Environmental Effects
In mammals, embryonic and fetal
development have a longer time course
-Thus they are more subject to the effects of
environmental contaminants, and bloodborne agents in the mother
-Thalidomide, a sedative drug
-Many pregnant women who took it
had children with limb defects
66
Environmental Effects
Endocrine disrupting chemicals (EDCs)
-Interfere with synthesis, transport or
receptor-binding of endogenous hormones
-Derived from three main sources
-Industrial wastes (polychlorinated
biphenyls or PCBs)
-Agricultural practices (DDT)
-Effluent of sewage-treatment plants
67