Bio 127 - Section IIIOrganogenesis Part 1

I. The Stem Cell Concept

II. The Emergence of the Ectoderm

III. Neural Crest Cells and Axonal Specificity

IV. 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

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

HSC

Maturational series of neuronal stem cells

VOCABULARY

c. Types: Embryonic Stem Cells

c. Types: Adult Stem Cells

• Committed stem cells with limited potential– hematopietic stem cells - hair stem cells– mesenchymal stem cells - melanocyte stem cells– epidermal stem cells - muscle stem cells– neural stem cells - tooth stem cells– gut stem cells - germline stem cells– mammary 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?

Hematopoietic stem cellscan form all blood cells.

Mesenchymal stem cellscan migrate to injury sites.

Controls on differentiation: -bone cell matrix -stromal paracrine factors -pericyte paracrine factors -systemic hormones -neuronal signals

So far.....Wntangiopoietinstem cell factorDelta-NotchIntegrin-ECM

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 themremain stem cells, while the daughters without contact become sperm progenitors

Hub cells Unpaired JAK-STAT Stem Cell Division

Stem cell niche in Drosophila testes

Cadherinsappear tohold firstcentrosomeclose 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 notochordcause neurulation in ectoderm above

Interestingly, the primary mechanism is by means of inhibition....

Figure 9.1 Major derivatives of the ectoderm germ layer

EctodermalCompetencies

DifferentiatedPhenotypes

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

PrimaryNeurulation

SecondaryNeurulation

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

Neural plate cells elongateinto columnar epithelium

As much as half of the ectodermcan be induced to form neural plate!

neural convergent extensioncombined with epidermal epiboly

medial hinge point cellsare anchored to notochord

MHP cells flatten and become wedge-shapedto facilitate bending

Primary Neurulation in the Chick

dorsolateral hinge pointsform between neural and epidermal cells, not crest

as the tube nears closure,neural crest cells undergoEMT and migrate away

*remember: closureresults from neural cells

switching from E- to N-cadherin

Birds close at mid-brain and “zip” in 2 directionsMammals have three primary points of closure

Works the same on the dorsal surface of amphibian “sphere”

Human Neural Closure

-spina bifida: posterior neuropore-anencephaly: anterior neuropore-craniorachischisis: the whole tube

Neural tube defects are common: 1 in 1000 live births

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 teratogenfumonisin 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

r1

r2

r3

r4

r5

r6r7

Neural crest cells from above specific rhombomeres form the cranial nerve ganglia

5th trigeminal

7th facial and8th vestibuloacoustic

9th glossopharyngeal

The size of the vertebrate brain increases very rapidly in early neurulationdue 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 manyneurons are able to ultimately divide and form

Occlusion of the neural tube allows expansion of the future brain

Relaxesafterexpansion

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 transcription factors

Pax7

Pax6

Nkx6.1

TGF-B

Shh

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!

can be 2-3 feet long

microtubules followssignal

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 ademyelination disease

Neural stem cells in the germinal epithelium

Neural tubestart as onelayer ofstem cells,all in the cell cycle

Position of nucleusdepends on cell cycle

Stem cell divisionsare 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 basiclayers are formed

stemcells

cellbodies“graymatter”

myelinaxons“whitematter”

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 gliaprovide themigratoryprocesses inthe cerebellum

phenomenaldual-photonconfocalmicroscopy!

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 fromdiencephalon, contacts ectoderm

2. Optic vesicle Delta binds to ectoderm Notch, induces placode

The induction of lens causes the cells to elongate, invaginate and grab holdof 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 opticcup to form pigmented and neural retina

4. Lens tissue is pulled under the surface, induced to make Crystallin

Key Cell Differentiation Events

Without theexpression of the retinalhomeoboxgene (Rx) nooccular tissuesdevelop at all

Key Cell Differentiation Events

The neural retinaforms 7 majorlayers of neurons

The epithelium ofthe posterior layerof optic cup arecompetent to makeall 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 neuralretina form the ciliary body, which secretesthe vitreous humor tocontrol pressure andthe curvature of eye.

Key Cell Differentiation Events

surfaceectoderm

Neural crest

“Lens fibers” are elongated cells from the lens placode

Neural crest mesenchymemigrate in to form cornea

Key Cell Differentiation Events

Anterior chamber fillswith vitreous humor

Neural crest cell MET, dehydrate andform tight junctions to become cornea.Stem cell population at corneal edge.

Lens fibers extrudetheir nuclei and formmassive amounts ofCrystallin proteins.Stem cells in epithelium.

Figure 9.31 Sonic hedgehog separates the eye field into bilateral fields

Too little Shh and the eyefields don’t separate on midline

“Cyclopism”

Figure 9.32 Surface-dwelling (A) and cave-dwelling (B) Mexican tetras (Astyanax mexicanus)

Too much Shh andeye fields don’t form.

Vision sacrificed incave dwellers in favorof better olfaction andbigger 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

The same basic design asthe neuroepithelium: stemcell divisions are horizontal,differentiation divisions arevertical.

Melanocytes transfer melanin granulesdirectly to the cells of the Malpighian layer.

Daughters migrate away,withdraw from cell cycle,differentiate to keratinocytes

Outerlayeris deadcells

Innerlayerdividesthroughlifetime

Layers of the human epidermis

Keratinocytes are the epitomyof “taking one for the team”!

They then arrest their own metabolismand die, leaving a tough membrane andfiber protective layer over the basal layer.

As they differentiate, theyexpress heavy keratin fibersin their cytoplasm, hook themto cadherins and integrins intheir plasma membrane andto collagen and neighbor cells.

Outerlayeris deadcells

Innerlayerdividesthroughlifetime

• 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 induceplacode formation in basal cells

2. Placode cells induce thosefibroblasts to form dermal papilla

placodesinksinto

dermis

Reciprocal induction in development of hair follicles

3. dermal papilla induces stemcells 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 thefollicle 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 fromthe epithelial cells releasing both Wnt10AND its inhibitor Dickkopf. Wnt causesautocrine formation of placodes close by,while Dickkopf blocks nearby neighborsfrom being able to form them.