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Cellular Mechanismsof Development
Chapter 19
2
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
3
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 cyclin-dependent kinases (Cdks)
During cleavage, the zygote is divided into smaller & smaller cells called blastomeres
-Moreover, the G1 and G2 phases are shortened or eliminated
4
Cell Division
5
Cell Division
CyclinDegradation
CM
G2
interphase
mitosis
cytokinesis
G2
SG1
M
C
Mitosis
DNA Synthesis
Adult Cell Cycle
S
G1 Cdk /G1cyclin
Cdk /S cyclin
Mitosis
DNA Synthesis
Active
Cell Cycle of Early Frog Blastomere
Active
Active
CyclinSynthesis
Active Cdk /G2
cyclinC
M
SS
M
Cdk /cyclin
Cdk
Inactive
a. b.
6
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
7
Nematode Lineage Map
a.
b.
Egg andsperm line
Egg
Pharynx
Cuticle-making cells Vulva
Egg Sperm
Adult Nematode
Vulva
GonadGonad
Gonad
Nervous system
Pharynx
Intestine
Cuticle
IntestineNervoussystem
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
8
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
9
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)
10
Egg
Sperm Blastocyst Embryo
Embryonic stem-cellculture
Inner cellmass
Once sperm cell and egg cell have joined, cellcleavage produces a blastocyst. The inner cell massof the blastocyst develops into the human embryo.
Embryonic stem cells (ES cells) areisolated from the inner cell mass
11
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
12
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
13
Donor No donor
RecipientBefore Overt
Differentiation
RecipientAfter Overt
Differentiation
Normal Not Determined(early development)
Determined(later development)
Tail cells aretransplanted
to head
Tail cells developinto head cells in head
Tail cells developinto tail cells in head
Tail cells aretransplantedto head
Tail Head
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
14
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
15
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
16
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
a.
n
2n
Embryo(diploid) 2n
Larva(diploid) 2n
Sperm(haploid) n
Egg(haploid) n
Pigmentgranules
Adult tunicate(diploid) 2n
ME
IOS
IS
MEIOSIS
METAMORPHOSIS
17
Cell Differentiation
Cytoplasmic determinants
-Female parent provides egg with macho-1 mRNA
-Encodes a transcription factor that can activate expression of muscle- specific genes
18
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
19
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
20
Anterior
Posterior
Anterior Posterior
a.
b. c.
21
FGF signaling
Sagittal section Longitudinal section
Longitudinal section
Dorsal nerve cord (NC)
Mesenchymal cells (Mes)
Tail muscle cells (Mus)
32-Cell Stage 64-Cell Stage
Anterior
Posterior
2
1
Notochord (Not)Ventral endoderm (En)
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
21
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
22
Cell Differentiation
a.
FGF Signal received?Mesenchyme
MuscleNotochord
FGF Signal received?
Macho-1 inherited?
Nerve cord
First Step Cell TypesSecond Step
YesNo
YesNo
Yes
No
FGF
FGF Receptor
Ras/MAPKPathway
T-Ets Macho-1
Cell membrane
P
MesenchymePrecursor Cells
Suppression of musclegenes and activation
of mesenchyme genes
b.
23
Cell DifferentiationNo
FGFFGF Receptor
Ras/MAPKPathway
T-Ets
Cell membrane
Nerve cordPrecursor Cells
FGF
FGF Receptor
Transcriptionof notochord genes
P
Notochord Precursor Cells
NoFGF
FGF Receptor
Ras/MAPKPathway
Transcription of muscle genes
MusclePrecursor Cells
Suppression of notochordgenes and activationof nerve cord genes
No Macho-1No Macho-1Macho-1
Cell membraneCell membrane
T-EtsT-Ets
Ras/MAPKPathway
24
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:
25
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
26
Development Implantation Birth of Clone Growth to Adulthood
Embryo begins todevelop in vitro.
Embryo isimplanted into
surrogatemother.
After a five-month pregnancy, alamb genetically identical to thesheep from which the mammarycell was extracted is born.
Embryo
Preparation Cell Fusion Cell Division
Mammary cell is extracted and grown in nutrient-deficient solution that arrests the cell cycle.
Egg cell is extracted. Nucleus is removed fromegg cell with a micropipette.
Nucleus containingsource
Mammary cell isinserted inside
covering of egg cell.
Electric shock fuses cellmembranes and triggers
cell division.
27
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
28
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
29
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
30
Cloning
The skin cellnucleus is insertedinto the enucleated
human egg cell.
Cell cleavageoccurs as the
embryo begins todevelop in vitro.
The embryoreaches the
blastocyst stage
The nucleus from a skin cell of a diabeticpatient is removed.
The nucleus from a skin cell of a healthy patient is removed.
Early embryo Blastocyst
Inner cellmass
EScells
Diabeticpatient
Healthypatient
31
CloningTherapeutic Cloning
Reproductive Cloning
Embryonic stem cells(ES cells) are extractedand grown in culture.
The blastocyst is kept intact andis implanted into the uterus of a
surrogate mother.
The resulting baby isa clone of the
healthy patient.
The stem cells are developedinto healthy pancreatic islet cells
needed by the patient.
The healthy tissue isinjected or transplantedinto the diabetic patient.
Healthy pancreatic islet cells
Diabeticpatient
32
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
33
Cloning
Early reports on a variety of adult stem cells indicated that they may be pluripotent
-Since then these results have been challenged
34
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
35
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
36
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
37
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
38
c.
b.
Nursecells
AnteriorPosterior
Movement ofmaternal mRNA
Oocyte
Folliclecells
Fertilized egg
a.
d.
e.
Three larval stages
Syncytial blastoderm
Cellular blastoderm
Nuclei line up alongsurface, and membranesgrow between them toform a cellular blastoderm.
Segmented embryo prior to hatching
Metamorphosis
Abdomen
Thorax
Head
Hatching larva
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Nucleus
Embryo
39
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
40
About 21/2 hours after fertilization, bicoid protein turns on a series of brief signals from so-called 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.
Forming the SegmentsLaying Down the Fundamental Regions
Setting the Stage for Segmentation
Bicoid
Hairy
Krüppel Hunchback
Engrailed
Establishing the Polarity of the Embryo
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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).
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 pair-rule gene hairy . Some pair-rule genes are only required for even-numbered segments while others are only required for odd numbered segments.
The final stage of segmentation occurs when a “segment- polarity” 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).
41
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
42
a.
b.
bicoid mRNA movestoward anterior end
bicoidmRNA
Movement ofmaternal mRNA
Nucleus
Microtubules
Nursecells
Folliclecells
nanos mRNA movestoward posterior end
PosteriorAnterior
PosteriorAnterior
nanosmRNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
43
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
44
Co
nce
ntr
atio
n
Anterior
Anterior
a. Oocyte mRNAs
c. Early cleavage embryo proteins
b. After fertilization
Posterior
Posterior
nanos mRNA
hunchback mRNA
bicoid mRNA
caudal mRNA
Nanos protein
Hunchback protein
Bicoid protein
Caudal proteinC
on
cen
trat
ion
Anterior Posterior
nanos mRNAhunchback mRNAbicoid mRNAcaudal mRNA
Nanos proteinHunchback proteinBicoid proteinCaudal protein
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
45
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
46
47
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
48
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
49
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
50
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
51
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
52
Production of Body Plan
Drosophila HOM genes
Thorax
Antennapedia complex
Head Abdomen
Bithorax complex
Fruit fly
Fruit flyembryo
Mouse
Hox 1
Hox 2
Hox 3
Hox 4
Mouseembryo
a. b.
Drosophila HOM Chromosomes Mouse Hox Chromosomes
lab pb Dfd Scr Antp Ubxabd-Aabd-B
53
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
54
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
55
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
56
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
57
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
58
a. b.
InhibitorCED-9 Bcl-2
CED-4 Apaf1
Caspase-8 or -9
Apoptosis
CED-3
Apoptosis
Inhibitor:
Activator:
ApoptoticProtease:
Caenorhabditis elegans Mammalian CellOrganism
Inhibition
Activation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
59
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)
60
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
61
Development of Seed Plants
2. Embryonic tissue formation-Three basic tissues differentiate:
-Epidermal, ground and vascular3. Seed formation
-1-2 cotyledons form-Development is arrested
4. Seed germination-Development resumes -Roots extend down, and shoots up
62
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
63
a. Early cell division
Embryo
Embryo
Suspensor
b. Tissue formation
d. Germinationc. Seed formation meristem
Seed wall
Shoot apicalmeristem
Root apical
Shoot apical meristem
Cotyledons
e. Meristematic development and morphogenesis
Root apicalmeristem
Epidermalcells
Groundtissue cellsVasculartissue cells
Cotyledons
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
64
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
65
Environmental Effects
66
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 blood-borne agents in the mother
-Thalidomide, a sedative drug
-Many pregnant women who took it had children with limb defects
67
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
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