Download Differentiation

Bio 127 - Section III
Organogenesis Part 1
I.
II.
III.
IV.
The Stem Cell Concept
The Emergence of the Ectoderm
Neural Crest Cells and Axonal Specificity
Paraxial and Intermediate Mesoderm
I. Stem Cells Role in the Development
of Tissues and Organs
• Gastrulation produces the three germ layers
• Germ layer interactions induce organogenesis
• More and more we see that this requires the
development of stem cells and their ‘niches’
– Places that these cells can remain relatively
undifferentiated and yet provide differentiated progeny
A. The Stem Cell Concept
• Division of stem cells produces one new stem
cell and one differentiated daughter
– Sometimes potential is unrealized and you get two
new stem cells
• In some organs: frequent replenishing divisions
– gut, epidermis, bone marrow
– example: billions of blood cells are destroyed by the
spleen every hour
• In others, they only divide in response to stress
or the need to repair the organ
– heart, prostate
b. Stem Cell Terminology
HSC
Totipotent = zygote and 4-8 blastomeres
Pluripotent = inner cell mass, “ESC”
COMMITTED STEM CELLS:
Multipotent = adult stem cells
hematopoietic, mammary, gut
Unipotent = adult stem cells
spermatogonia, melanocyte
Maturational series of neuronal stem cells
V
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c. Types: Embryonic Stem Cells
c. Types: Adult Stem Cells
• Committed stem cells with limited potential
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hematopietic stem cells
mesenchymal stem cells
epidermal stem cells
neural stem cells
gut stem cells
mammary stem cells
- hair stem cells
- melanocyte stem cells
- muscle stem cells
- tooth stem cells
- germline stem cells
• Hard to extract and culture
– HSC are less than 1 in 15,000 bone marrow cells
– Transplants work very well, however
– Mammary, neural, muscle, others all being worked on
c. Types: Mesenchymal Stem Cells
• Surprising degree of differentiation plasticity
- muscle, fat, bone, cartilage
- PDGF, TGF-B, FGF combinations determine fate
• Found in lots of niches in both embryo and adult
– umbilical cord blood, baby teeth
– marrow, fat muscle, thymus, dental pulp
• Paramedic response to injury
– Migrate from niche to provide paracrine stimulus to
repair injured tissues w/wo differentiating on-site
2. The Stem Cell Niche
• Part of organogenesis in many tissues requires
developing special sites for stem cells to live
• Microenvironments wherein the cells that stay
don’t differentiate but those that leave do
• Unique combinations of local paracrine
signaling, cell-ECM and cell-cell interactions
Hematopoietic stem cells and the bone marrow niche
-Both are Committed Stem Cells
-Progenitor cells can’t self-renew
This is what allows us to do bone marrow transplants
So, what’s going on in the bone marrow niche?
Controls on differentiation:
-bone cell matrix
-stromal paracrine factors
-pericyte paracrine factors
-systemic hormones
-neuronal signals
So far.....
Wnt
angiopoietin
stem cell factor
Delta-Notch
Integrin-ECM
Hematopoietic stem cells
can form all blood cells.
Mesenchymal stem cells
can migrate to injury sites.
The mouse tooth stem cell niche (we don’t have one)
A balance of “positive – negative”
FGF3 – BMP4 and activin -- follistatin
Stem cell niche in Drosophila testes
The ‘hub’ consists of ~12 somatic cells: the cells in direct contact with them
remain stem cells, while the daughters without contact become sperm progenitors
Hub cells  Unpaired  JAK-STAT  Stem Cell Division
Stem cell niche in Drosophila testes
Cadherins
appear to
hold first
centrosome
close to
the ‘hub’
Niche Break-Down May be Part of Aging
• Too much cell differentiation
– Can deplete the capacity for renewal
– Graying hair may result from too many
melanocyte differentiations
• Too much cell division
– Cancers may result from excess division
– Myeoloproliferative disease is too much
marrow division without differentiation
Neurulation is a developmental process
that takes the organism from the gastrula
stage through development of a functional
central nervous system
Structure  Process  Structure
The first organ system to begin development
in vertebrates is the central nervous system
Two Major Steps:
1. Formation of the neural tube
2. Differentiation of neurons
REMEMBER: Hensen’s Node (chick) and Spemann’s Organizer
(frog) pass organizing power to the notochord
Secreted factors from the notochord
cause neurulation in ectoderm above
Interestingly, the primary mechanism is by means of inhibition....
Figure 9.1 Major derivatives of the ectoderm germ layer
Ectodermal
Competencies
Differentiated
Phenotypes
So, where are we starting?
Establishing Neural Cells from the Ectoderm
• Competence: multipotent cells with the ability to
form neurons with the right signals
• Specification: the right signals are there but cell
change could still be repressed by other signals
• Determination: the cells have entered the
neuronal pathway and cannot be repressed
• Differentiation: the cells leave the mitotic cycle
and express the genes characteristic of neurons
As the node regresses, it leaves the notochord behind
anterior to posterior and the overlying neural plate starts
to form neural tube in the same pattern
Primary
Neurulation
Secondary
Neurulation
Combining Primary and Secondary
Neurulation to form the Neural Tube
• Primary = Folding of the Neural Plate into
a tube structure directly
• Secondary = Mesenchymal Coalescence
followed by hollowing out into a tube
• The Neural Tube proper results from the
joining of the two
• In Birds: everything anterior to the hind
limbs is Primary Neurulation
• In Mammals: the sacral vertebrae back
through the tail is Secondary Neurulation
• In Amphibians and Fish: only the tail is
Secondary Neurulation
Primary Neurulation in the Chick
As much as half of the ectoderm
can be induced to form neural plate!
Neural plate cells elongate
into columnar epithelium
neural convergent extension
combined with epidermal epiboly
medial hinge point cells
are anchored to notochord
MHP cells flatten and
become wedge-shaped
to facilitate bending
Primary Neurulation in the Chick
dorsolateral hinge points
form between neural and
epidermal cells, not crest
as the tube nears closure,
neural crest cells undergo
EMT and migrate away
Birds close at mid-brain and “zip” in 2 directions
Mammals have three primary points of closure
*remember: closure
results from neural cells
switching from E- to N-cadherin
Works the same on the dorsal surface of amphibian “sphere”
Human Neural Closure
Neural tube defects are common: 1 in 1000 live births
-spina bifida: posterior neuropore
-anencephaly: anterior neuropore
-craniorachischisis: the whole tube
Folate Supplementation Reduces Rate of Defects
Folate-binding protein in the neural
folds as neural tube closure occurs
A fungal contamination of corn produces the teratogen
fumonisin that appears to disrupt the function of FBP
Secondary Neurulation
The coalescence of the two neural
tubes is not well understood and
may be important in some defects
Differentiation of the Neural Tube
• Three simultaneous levels of development
– Gross anatomy: bulges and constrictions form the chambers of
the brain and spinal cord
– Tissue anatomy: the cell populations in the wall rearrange to
form functional domains
– Cell biology: the neuroepithelial cells differentiate into neurons
and glia
• Two simultaneous axes of development
– Anterior-Posterior: the forebrain back toward the spinal column
– Dorsal-Ventral: the axis from the roof plate of the tube, near the
epidermis, and the floor plate, near the notochord
Figure 9.9 Early human brain development (Part 1)
Figure 9.9 Early human brain development (Part 2)
Rhombomeres of the chick hindbrain
Neural crest cells from above specific
rhombomeres form the cranial nerve ganglia
r1
r2
5th trigeminal
r3
r4
7th facial and
8th vestibuloacoustic
r5
r6
r7
9th glossopharyngeal
The size of the vertebrate brain increases very rapidly in early neurulation
due to an osmotic Na+ gradient dumped into the presumptive ventricle:
for example, the chick’s brain volume increases 30-fold from day 3-5
The increase in size determines how many
neurons are able to ultimately divide and form
Occlusion of the neural tube allows expansion of the future brain
Relaxes
after
expansion
Anterior-Posterior Specification of Neurons: Evolutionary
conservation of homeotic gene organization and transcriptional
expression in fruit flies and mice
Dorsal-ventral specification of the spinal neural tube
Dorsal-ventral specification of the spinal neural tube
Sensory Input
Motor Output
Concentration Gradient-Dependent Transcription Factor Expression
growth factors
TGF-B
transcription factors
Pax7
Pax6
Shh
Nkx6.1
Differentiation of Neurons in the Brain
• Neuroepithelium of neural tube starts as one
layer of stem cells
• Humans have 100 billion neurons and 1 trillion
glial cells
• Neuroepithelium gives rise to:
– Ependymal cells: line the ventricles, secrete CSF
– Neurons: electrical, regulation, thought, senses
– Glia: brain construction, neuron support, insulation
and maybe memory storage?
Diagram of a neuron
We have very few dendrites at birth,
up to 100,000 connections in 1st year!
microtubules
can be 2-3 feet long
follows
signal
gradient
Figure 9.16 Axon growth cones
Actin microspikes provide migratory traction and signal sensing
Figure 9.17 Myelination in the central and peripheral nervous systems
Multiple sclerosis is a
demyelination disease
Neural stem cells in the germinal epithelium
Neural tube
start as one
layer of
stem cells,
all in the
cell cycle
Position of nucleus
depends on cell cycle
Stem cell divisions
are all horizontal
Neuron Birthdays
• Differentiating cells are born from vertical divisions
• Stem cell stays attached, distal sister migrates away and
leaves the cell cycle
• Early birthdays form closer layers, later birthdays form
more distal layers
• Neuronal function, neurotransmitter type and
connections formed depends on Anterior-Posterior,
Dorsal-Ventral position (eg. Hox, TGF-B v. Shh )
Complexity Increases the Further Anterior You Go
Initially three basic
layers are formed
stem
cells
cell
bodies
“gray
matter”
myelin
axons
“white
matter”
Figure 9.20 Development of the human spinal cord
Original formation of the germinal neuroepithelial layer
Differentiated three adult layers:
1. ventricular zone = ependyma
2. Intermediate zone = mantle
3. Marginal zone = myelin layer
Becomes encased in connective tissue
Figure 9.19 Differentiation of the walls of the neural tube (Part 1)
Differentiation of the Cerebellum
Adds three additional layers:
- The Purkinje cell layer
- The inner and outer granular layers
Cerebellum coordinates complex movements
- Purkinje’s have ~100,000 synapses on their dendrites
- Axons control all cerebellar output
- Not too sure about the role of granular neurons
Cerebellar neurons have typical brain migration mechanism
They crawl along glial processes from layer to layer
Figure 9.21 Cerebellar organization
Bergman glia
provide the
migratory
processes in
the cerebellum
phenomenal
dual-photon
confocal
microscopy!
Cerebral cortex is the most complex of all
The major addition is the formation of the neocortex
- Stratifies into 6 layers, each with unique inputs and outputs
- Adult form not completed until middle of childhood
Also organized Anterior-Posterior and Dorsal-Ventral
- Hox genes and TGF-B v. Shh
- eg. layer 6 inputs and outputs in visual cortex
differ from layer 6 connections in auditory cortex
Figure 9.25 Evidence of adult neural stem cells
Development of the Sensory Systems
• They form from the cranial ectodermal placodes
which are made competent by endo, meso signals
– We’ll focus on the lens placode
– The olfactory placode forms nasal epithelium, nerves
– The otic placode forms inner ear, acoustic ganglia
Reciprocal induction between neural tube and overlying ectoderm
1. Optic vesicle evaginates from
diencephalon, contacts ectoderm
2. Optic vesicle Delta binds to
ectoderm Notch, induces placode
The induction of lens causes the cells to elongate, invaginate and grab hold
of the optic vesicle cells with adhesive filopodia to ride its movement inward
Reciprocal induction between neural tube and overlying ectoderm
3. Lens signals cause two layers of optic
cup to form pigmented and neural retina
4. Lens tissue is pulled under the
surface, induced to make Crystallin
Key Cell Differentiation Events
Without the
expression
of the retinal
homeobox
gene (Rx) no
occular tissues
develop at all
Key Cell Differentiation Events
The neural retina
forms 7 major
layers of neurons
The epithelium of
the posterior layer
of optic cup are
competent to make
all of them
Key Cell Differentiation Events
The tips of the optic cup form a ring of pigmented muscle,
the iris, which controls the pupil dilation and gives eye color
The junction between
the iris and the neural
retina form the ciliary
body, which secretes
the vitreous humor to
control pressure and
the curvature of eye.
Key Cell Differentiation Events
“Lens fibers” are elongated
cells from the lens placode
surface
ectoderm
Neural crest
Neural crest mesenchyme
migrate in to form cornea
Key Cell Differentiation Events
Anterior chamber fills
with vitreous humor
Neural crest cell MET, dehydrate and
form tight junctions to become cornea.
Stem cell population at corneal edge.
Lens fibers extrude
their nuclei and form
massive amounts of
Crystallin proteins.
Stem cells in epithelium.
Figure 9.31 Sonic hedgehog separates the eye field into bilateral fields
Too little Shh and the eye
fields don’t separate on midline
“Cyclopism”
Figure 9.32 Surface-dwelling (A) and cave-dwelling (B) Mexican tetras (Astyanax mexicanus)
Too much Shh and
eye fields don’t form.
Vision sacrificed in
cave dwellers in favor
of better olfaction and
bigger jaws!
The Epidermis and Cutaneous Appendages
• Remember, the ectoderm forms:
– Neural tube
– Neural crest
– Epidermis
• The epidermis is the outer layer of the skin
with mesoderm-derived dermis underneath
– Largest organ, tough, impermeable, renewable
The Epidermis and Cutaneous Appendages
• The cells of the skin include
– Basal layer stem cells, keratinocyte daughters
– Dermal fibroblasts
– Neural crest-derived melanocytes
• The same three cell types form hair follicles
– Each must become specialized to do so
– Similar interactions form feathers and scales
Layers of the human epidermis
Outer
layer
is dead
cells
The same basic design as
the neuroepithelium: stem
cell divisions are horizontal,
differentiation divisions are
vertical.
Daughters migrate away,
withdraw from cell cycle,
differentiate to keratinocytes
Inner
layer
divides
through
lifetime
Melanocytes transfer melanin granules
directly to the cells of the Malpighian layer.
Layers of the human epidermis
Outer
layer
is dead
cells
Keratinocytes are the epitomy
of “taking one for the team”!
As they differentiate, they
express heavy keratin fibers
in their cytoplasm, hook them
to cadherins and integrins in
their plasma membrane and
to collagen and neighbor cells.
Inner
layer
divides
through
lifetime
They then arrest their own metabolism
and die, leaving a tough membrane and
fiber protective layer over the basal layer.
• Basal cells express Delta-family member Jagged
• When it binds to distal sister Notch it starts
keratinocyte differentiation
• Time from basal layer to sloughed is 2 weeks in
mouse, a little longer in us
• As part of their differentiation they push their
nucleus to the side of the cell
• We lose 1.5 grams of them per day with enough
DNA in each (or perhaps a few) to identify us!
Reciprocal induction in development of hair follicles
1. Dermal fibroblasts induce
placode formation in basal cells
placode
sinks
into
dermis
2. Placode cells induce those
fibroblasts to form dermal papilla
Reciprocal induction in development of hair follicles
3. dermal papilla induces stem
cells to differentiate daughters
differentiated daughters include:
hair shaft, sebocytes, root sheath
Part of differentiation includes migration to absorb papilla.
Reciprocal induction in development of hair follicles
- Hair shaft is keratinocytes with melanin just like skin itself.
- Sebaceous gland secretes lubricant onto hair and skin.
- “Bulge” is the stem cell niche for basal cells and melanocytes.
Figure 9.42 Model of follicle stem cell migration and differentiation
Stem cells migrate from the bulge: 1. down the outer root sheath to the
follicle root, 2. up to the sebaceous gland, and 3. up to the basal layer.
Figure 9.39 Patterning of hair follicle placodes by Wnt10 and Dickkopf
Evenly spaced hair follicles result from
the epithelial cells releasing both Wnt10
AND its inhibitor Dickkopf. Wnt causes
autocrine formation of placodes close by,
while Dickkopf blocks nearby neighbors
from being able to form them.