Chapter 18, 20, 47

EXCLUDE CHP 21
Intro
- During embryonic development: Fertilized egg gives rise to many different cell types
- Cells are organized successively into tissues, organs, organ systems & the whole
organism
- Gene expression orchestrates developmental programs of animals

Essential Developmental Processes (NOT IMPORTANT)

- A. Cell proliferation (cell division)
- Process resulting in increase of number of cells
- Defined by balance between cell divisions & cell loss through cell death or
differentiation
- Increased in tumors
- Don't have the balance
- B. Cell-Cell Interactions
- Direct interactions between cell surfaces
- Play crucial role in development & function of multicellular
organisms
- These interactions allow cells to communicate with each other in response to
changes in their microenvironment
- Cell-Cell Recognition
- Animal cells may communicate by direct contact
- Important in embryonic development & immune response
- C. Cell Differentiation
- Creating cells with different characteristics at different positions
- D. Cell Movement and Expansion
- Rearranging cells to form structured tissues & organs
- E. Apoptosis
- Death of cells which occurs as a normal & controlled part of an organism's
growth or development

Sources of Developmental Information

- Genes regulated differently in each cell type
- Differential gene expression
- How?
- A. Cytoplasmic Determinants
- Materials in egg set up a program of gene regulation
- Carried out as cells divide
- B. Inductive Signals
- A. Cytoplasmic Determinants
- In unfertilized egg cytoplasm:
- RNA
- Proteins
- Organelles
- Other substances
- Distributed unevenly
- Cytoplasmic determinants = Maternal substances in egg that influence early
development
- When the zygote starts dividing, these substances determine differential
gene expression
- As zygote divides by mitosis, cells contain different cytoplasmic determinants
- Lead to different gene expression
- B. Inductive Signals
- Other major source of developmental information is the environment around cell,
especially signals from nearby embryonic cells
- In the process called induction, signal molecules from embryonic cells cause
changes in nearby target cells
- Interactions between cells induce differentiation of specialized cell types
- Sequential Regulation of Gene Expression During Cellular Differentiation
- Determination = Irreversibly commitment of a cell to becoming a particular cell
type
- Determination precedes differentiation
- Differentiation = Process by which cells become specialized in structure &
function
- Cell differentiation is marked by the production of tissue-specific proteins
- Only found in specific cell types
- Provide cell with its characteristic structure & function
- EX:
- Myoblasts are cells determined to form muscle cells
- Produce large amounts of muscle-specific proteins
- MyoD is a “master regulatory gene” that encodes a transcription factor that
commits the cell to becoming skeletal muscle
- Some target genes for MyoD (protein) encode additional muscle-specific
transcription factors
- Determination
- Signals from other cells (i.e., induction) lead to activation of a
master regulatory gene called myoD
- Cell makes MyoD protein (transcription factor activator)
- Now a myoblast, the cell is irreversibly committed to becoming a
skeletal muscle cell
 - Differentiation
- MyoD protein stimulates the myoD gene further (positive feedback)
- Activates genes encoding other muscle-specific transcription factors
- Activate genes for muscle proteins
- Turns on genes that block the cell cycle, thus stopping cell division
- Nondividing myoblast fuse to become mature multinucleated muscle cells
= muscle fibers
- Pattern Formation: setting up the body plan
- Pattern formation = Development of a spatial organization of tissues &
organs
- In animals, pattern formation begins with establishment of major axes
- Dorsal, anterior, right, left, posterior, ventral
- Positional information = Molecular cues that control pattern formation

- Tells a cell its location relative to body axes & to neighboring cells
- Pattern formation has been extensively studied in fruit fly Drosophila
melanogaster
- Combination of anatomical, genetic & biochemical approaches
- Researchers have discovered developmental principles common to many
other species, including humans
- Life Cycle of Drosophila
- In Drosophila, cytoplasmic determinants in the unfertilized egg determine
the axes before fertilization
- After fertilization, the embryo develops into a segmented larva with three
larval stages
- The larva then forms a pupa, which undergoes metamorphosis into adult
fly
- 1. Egg cell with nucleus, follicle cells and nurse cells (support cells)
- 2. Nurse cells shrink and give materials to the growing egg shell
- 3. Egg is fertilized, then gets laid outside
- 4. Segmentation pattern forms by creating a larva
- 5. Eventually the larva is formed with it’s right and left axis
- Genetic Analysis of Early Development
- Edward Lewis, Christiane Nüsslein-Volhard & Eric Wieschaus won a Nobel Prize
in 1995 for decoding pattern formation in Drosophila
- Abnormal Pattern Formation in Drosophila
- Lewis discovered homeotic genes by studying bizarre mutant flies with
developmental defects (e.g., extra wings, legs in wrong place)
- Located the mutations on the fly’s genetic map
- Connected developmental abnormalities to specific genes
 - Homeotic genes = Genes that control pattern formation in the late
embryo, larva & adult stages
- Mutations result in transformations in the identity of entire body parts
- Genetic Analysis of Segment Formation
- Nüsslein-Volhard & Wieschaus studied segment formation
- They created mutants, conducted breeding experiments & looked for
corresponding genes
- Many of the identified mutations were embryonic lethals, causing death
during embryogenesis
- They found ~120 genes essential for normal segmentation
- Axis Establishment
- Maternal effect genes = Encode cytoplasmic determinants that initially establish
the body axes of Drosophila
- Also called egg-polarity genes
- They control orientation of the egg & consequently the fly
- When maternal effect genes are mutant in mother
- Mutant phenotype in offspring, regardless of offspring’s own genotype
- Even if the offspring does not have the mutation, it shows the phenotype
- Phenotype = Observable physical & physiological traits of an organism, which
are determined by its genetic make-up
- Genotype = Genetic make-up of an organism
- EX: Bicoid
- One maternal effect gene, the bicoid gene, affects the front half of the
body (head)
- An embryo whose mother has no functional bicoid gene lacks the front
half of its body & has duplicate posterior structures at both ends
- Bicoid means “two-tailed”
- If the mom has a mutated bicoid gene, the offspring will have two
tails even if its DNA is not mutated
- This two-tailed phenotype suggests that the product of the mother’s bicoid
gene is essential for setting up the anterior end (head) of the embryo
- Bicoid Morphogen Gradient Hypothesis
- This hypothesis (i.e., product of mother’s bicoid gene being essential for
proper anterior-posterior axis set up) is an example of the morphogen
gradient hypothesis
- Gradients of substances called morphogens (e.g., bicoid mRNA/protein)
establish an embryo’s axes & other features of its form
- Experiments showed that bicoid protein is distributed in an anterior to
posterior gradient in the early embryo
- The bicoid proteins are distributed along the egg as a gradient and
determine polarity of the egg
 - The head will develop in areas of high concentration of bicoid
- The tail will develop in areas of low concentration of bicoid
- Bicoid Research
- The bicoid research was groundbreaking for three reasons:
- It identified a specific protein required for some early steps in pattern
formation (bicoid protein)
- It increased understanding of the mother’s role in embryo development
- It demonstrated that a gradient of molecules can determine polarity &
position in the embryo

Differential Gene Expression

- Organismal Cloning
- Produces one or more organisms genetically identical to the “parent” that donated
a single cell
- Different from gene cloning & cell cloning (e.g., via asexual reproduction)
- Interest comes primarily from its ability to generate stem cells
- Stems cells: great potential to regenerate damaged tissue
- Cloning Animals: Nuclear Transplantation
- Nuclear transplantation
- Nucleus of unfertilized egg cell or zygote is replaced with nucleus of a
differentiated cell
- Or: Somatic Cell Nuclear Transfer
- 1. Have somatic cells and egg cell and remove the nucleus from both
- 2. Add nucleus of the somatic cell to the egg cell

- A. Nuclear Transplantation in Frogs

- Experiments with frog embryos have shown that a transplanted nucleus can often
support normal development of the egg
- However, the older the donor nucleus, the lower the percentage of normally
developing tadpoles
 - As cells become more differentiated, changes happen in the nucleus
- 1. Remove nucleus of frog egg cell by exposing cell to UV light
- 2. Put nucleus of frog embryo cell (less differentiated) into the frog egg cell
- 3. Developed into Tadpoles
- 2. Put a nucleus from a fully differentiated cell from a tadpole into the egg cell
- 3. Most stop developing
- B. Nuclear Transplantation in Mammals
- Cloning of Dolly
- In 1997, Scottish researchers announced the birth of Dolly (first cloned
mammal)
- Dolly = Lamb cloned from an adult sheep by nuclear transplantation from
a differentiated mammary cell
- 1. Took egg cell from black face sheep and removed nucleus
- 2. Put nucleus into somatic cell of Finn-Dorset sheep
- 3. Allowed cell to divide and grow then put it into surrogate
- 4. Dolly was genetically identical to the Finn Dorset
- Dolly’s premature death in 2003, as well as that of another cloned sheep
from another experiment, led to speculation that her cells were not as
healthy as those of a normal sheep
- This possibly reflects incomplete reprogramming of original transplanted
nucleus
- Cloning of CC
- Since 1997, cloning has been demonstrated in many mammals, including
mice, cats, cows, horses, mules, pigs, monkeys & dogs
- CC (for Carbon Copy) was the first cat cloned
- However, CC differed somewhat from her female “parent”
- Had different fur color than the parent
- Due to random X chromosome inactivation (normal occurrence
during embryonic development)
- Cloned animals do not always look or behave exactly the same
- Even identical human twins, natural “clones,” are always slightly different
- Environmental influences & random phenomena play a significant role
during development
- Faulty Gene Regulation in Cloned Animals
- In most nuclear transplantation studies, only a small percentage of cloned
embryos have developed normally to birth
- Many cloned animals exhibit defects (e.g., obesity, pneumonia, premature
death)
- Nuclei of fully differentiated cells:
- Small subset of genes turned on
- Rest turned off due to epigenetic changes
 - Acetylation of histones
- Methylation of DNA
- Many epigenetic changes must be reversed in nucleus from a donor
animal in order for genes to be expressed or repressed
appropriately for early stages of development
- Success of cloning depends largely on modifying the chromatin in donor
nucleus to resemble that of a newly fertilized egg
- C. Stem Cells
- Stem Cells
- Relatively unspecialized cells that can both reproduce indefinitely and
under certain conditions, differentiate into one or more specialized cell
types
- Replenish stem cells
- Generate differentiated cells
- Embryonic and Adult Stem Cells
- Many early embryos (i.e., blastula stage) contain stem cells capable of
giving rise to differentiated embryonic cells of any type
- In culture, these embryonic stem (ES) cells reproduce indefinitely
- Depending on culture conditions, they can be made to differentiate into a
variety of specialized cells
- Adult stem cells can generate multiple (but not all) cell types
- They are used in the body to replace non reproducing cells as
needed
- Adult stem cells
- Small amount
- Can be found in
- Bone marrow - different kind of blood cells
- Peripheral blood
- Brain - Certain kinds of nerve cells
- Spinal cord
- Dental pulp
- Blood vessels
- Skeletal muscle
- Epithelia of the skin
- Epithelia of the digestive system
- Cornea
- Retina
- Liver
- Pancreas
- Embryonic stem cells
- Embryonic stem cells are pluripotent
 - Capable of differentiating into many different cell types
- Originally, ES cells were obtained only from embryos donated by patients
undergoing infertility treatments
- The techniques for cloning human embryos are still being optimized, but
they represent a potential new (less controversial) source of ES cells
- The main aim of cloning ES cells is to produce cells for treating disease
- Certain kinds of brain cells (Parkinson’s & Huntington’s disease)
- The process is thus called therapeutic cloning
- Opinions vary about the morality of therapeutic cloning

Model Organisms
- Biologists use model organisms to study development, chosen for the ease with which
they can be studied in the laboratory
- Development occurs at many points in the life cycle of an animal
- Across a range of animal species, embryonic development involves common stages that
occur in a set order
- Set Order of Embryonic Development
- 1. Fertilization
- 2. Cleavage
- 3. Gastrulation
- 4. Organogenesis
- 5. Morphogenesis
- Gastrulation and Organogenesis
- Origin and development of morphological traits
- Characteristics of Model Organisms
- Development similar to larger group of organisms
- Short generation time
- Relatively small genome
- Well characterized in terms of its genes & overall development

Development in Diverse Animals

- Fruit Flies (Insects – Arthropods)
- Sea Urchins (Echinoderms)
- Fishes (Teleost)
- Frogs (Amphibians)
- Chicks (Birds)
- Humans (Mammals)
- 1. Phase - Fertilization
- Formation of a diploid zygote from haploid egg & haploid sperm
- Molecules & events at egg surface play crucial role in each step of fertilization:
- Sperm penetrate the protective layer around the egg
 - Receptors on egg surface bind to molecules on sperm surface
- Changes at egg surface prevent polyspermy
- Entry of multiple sperm nuclei into the egg
- Polyspermy would lead to abnormal number of chromosomes
- Lethal to embryo
- The Acrosomal Reaction
- The acrosomal reaction is triggered when the sperm meets the egg
- The acrosome at the tip of the sperm releases hydrolytic enzymes that digest
material surrounding the egg
- Proteins at acrosomal process bind to specific receptor proteins
- “Lock-and-key”
- Ensures same species
- Especially important in external fertilization
- Contact & fusion of sperm & egg membranes
- Triggers depolarization of egg membrane (Na+ ions diffuse into egg)
- Sets up fast block to polyspermy
- Multiple sperms cannot get in the egg because of the sodium
channels
- Initiates the cortical reaction

- The Cortical Reaction

- Seconds after the sperm binds to the egg, vesicles just beneath the egg plasma
membrane release their contents & form a fertilization envelope
- The fertilization envelope acts as a slow block to polyspermy
 - The cortical reaction requires a high concentration of calcium (Ca2+) ions in the
egg
- A change in Ca2+ concentration seems to trigger the cortical reaction:
- Ca2+ spread across the egg correlates with the appearance of the
fertilization envelope
- Egg Activation
- Rise in Ca2+ in cytosol increases rates of cellular respiration & protein synthesis
by egg cell
- With these rapid changes in metabolism, the egg is said to be activated (fertilized)
- The proteins & mRNAs needed for activation are already present in egg (i.e.,
cytoplasmic determinants)
- Sperm nucleus fuses with egg nucleus - Cell division begins (marks end of
fertilization phase)
- Fertilization in Humans
- Fertilization in mammals & other terrestrial animals is internal
- A sperm must travel through a layer of follicle cells surrounding the egg before it
reaches the zona pellucida, or extracellular matrix of egg
- Sperm binding triggers cortical reaction
- In mammals, the first cell division occurs 12–36 hours after sperm binding [~1.5
hours in urchins]

- 2. Phase - Cleavage
- Fertilization is followed by cleavage, a period of rapid cell division without
growth
- Cleavage partitions the cytoplasm of one large cell into many smaller cells called
blastomeres
- Blastula = Ball of cells with a fluid-filled cavity called a blastocoel
- Cleavage in Frog Embryos
- In frogs & many other land animals, cleavage is asymmetrical due to
distribution of yolk (stored nutrients)
- The vegetal pole has more yolk; the animal pole has less yolk
- The yolk greatly affects the pattern of cleavage
- The first two cleavage furrows in the frog form four equally sized
blastomere
- The third cleavage is asymmetrical, forming unequally sized blastomeres
- This asymmetry is due to the yolk in the vegetal hemisphere

- Cleavage Patterns in Other Animals

 - Holoblastic cleavage = Complete division of the egg
- Occurs in species whose eggs have little or moderate amounts of
yolk, such as sea urchins & frogs
- Meroblastic cleavage = Incomplete division of the egg
- Occurs in species with yolk-rich eggs, such as reptiles & birds
- In Drosophila & other insects, multiple rounds of mitosis occur without
cytokinesis
- Morphogenesis
- Morphogenesis = Process by which cells occupy their appropriate
locations, involves:
- Gastrulation = Movement of cells from blastula surface to interior
of embryo
- Organogenesis = Formation of organs
- 3. Phase - Gastrulation
- Gastrulation rearranges the cells of a blastula into a two- or three-layered
embryo called a gastrula
- The three layers produced by gastrulation are called embryonic germ layers:
- Ectoderm forms the outer layer
- Endoderm lines the digestive tract
- Mesoderm partly fills the space between endoderm & ectoderm
- Each germ layer contributes to specific structures in the adult animal
- Germ Layers in Vertebrates
- Ectoderm (outer most layer)
- Epidermis of skin
- Nervous and sensor systems
- Pituitary gland
- Jaws and teeth
- Mesoderm (middle layer)
- Skeletal and muscular system
- Circulatory and lymphatic system
- Excretory and reproductive
- Dermis of skin
- Endoderm
- Epithelial lining of digestive tract
- Epithelial lining of respiratory, excretory, and reproductive tracts
and ducts
- Thymus, thyroid, and parathyroid glands
- When the blastula has two openings, it becomes a gastrula
- Gastrulation in other animals
- Diploblast has two germ layers
- Triploblast has three germ layers
- First blastopore is the mouth, second is the anus for Planaria (protostome)
- First blastopore is anus, second is mouth for Sea urchin (deuterostome)

- Gastrulation in Frogs
- Frog gastrulation begins when a group of cells on the dorsal side of the
blastula begins to invaginate
- This forms a crease along the region where the gray crescent formed
- Cells continue to move from the embryo surface into the embryo by
involution
- These cells become the endoderm & mesoderm
- Cells on the embryo surface will form the ectoderm
- The newly formed cavity is called the archenteron
 - This opens through the blastopore, which will become the anus
(i.e., deuterostomes: “mouth second”)
- Gastrulation in Chicks/Birds
- Prior to gastrulation, the embryo is composed of an upper & lower layer,
the epiblast & hypoblast, respectively
- Hypoblast is the invisible layer that surrounds the yolk and keeps it
together
- During gastrulation, epiblast cells move toward the midline of the
blastoderm
- Then into embryo toward yolk
- Midline thickens “Primitive streak”
- Hypoblast cells contribute to
- sac that surrounds yolk
- connection between yolk & embryo
- but do not contribute to embryo itself

Take Home
- Sources of Developmental Information
- Cytoplasmic Determinants
- Induction
- Sequential Gene Regulation: Determination vs. Differentiation
- Pattern Formation
- Organismal Cloning
- Nuclear Transplantation
- Stem Cells
- Order & Process of embryonic development
- Fertilization
- Cleavage
- Gastrulation
-