1. Initial rostrocaudal body location, which dictates initial Nc cell fate and alters which cellular and molecular signaling pathways are operational 

2. initial diffusible factors -- expressed at different developmental times, in a concentration -dependent manner, and in different combinations result in differential specification of ectodermal, neural tube and NC cell phenotypes

3.different genetic (transcription factors) and epigenetic mechanisms, which contribute to cell fae, as well as start cell delamination and migration 

4. different cell-cell interactions and migratory paths through the body can shape different NC phenotypes

5. The same "guidance" protein can repulse some migratig NC cells while attracting others and these proteins and teir receptors can change their expression levels in different regions of the body and over time. 

6. Target tissue cells can provide chemoattractant and stop signals for migration and trigger terminal differentiation of NC cells 

7. Slightly differing factors in different vertebrate species. 

-NC cells forms a the border between epidermal and neural ectoderm

- NC cells are highly migratory and form most of the PNS and several other tssues

1.  Chimericanimals(e.g.chick-quailchimera)

2.  Cell lineage analysis using dyes & cell-specificantibodies

PNS neuronal cell derivatives (including all PNS glial cells) 

- also some non-neural cells 

motor neurons, muscles cells, and CNS cells

- muscles that start and end inside the nervous sytem does not originate from the neural crest cells. 

- are pluripotential and any NC stem cell from any level of early neuraxis can take a new identity (different fate) when transplanted to a new location. 

- some tissues that arise from neural crests are: 

1. GI tract

2. pigment cells like skin tone 

3. cardiac cells 

4. bones and cartilage of face tissues at the early stages (mesodermal tissues are what directed the ectoderm into the neural tissues) 

feather colour is an outward phenotypic marker for transplanted neural crest cells from quail donor.

- this is called the quail-chick marker system. There are 2 ways for detecting quail from chick cells 

A. Feulgen staining of DNA shows a large mass of heterochromatin in the centre of the nucleus which is associated with the quail nucleolus. Heterochromatin is more evenly distributed in chick

- these cell nuclei are distinct so we can tell which cells belong to which 

s cell divides through mitosis. that particular marker will follow all the daughter cells. 

- can tell where a cell comes from and where its going

- can help tell what part of the neural crest in terms of rostrocaudal location becomes what. 

can now take any cell from the body and transform that into a progenitor cell and let it divide and differentiate into any cells. These progenitor cells (IPS cells) can be regenerated experimentally. 

ex. can take a hair cell and transform it and change it into neurons in the lab. A lot of the genes that we use for inducing progenitor cells from adult cells ar the same that are involved in cancer

-there's non genetic cancer approaches we can use, problems is that they are inefficient so transformed cells can take monthes. 

- there's no effiencient or effectives since takes too long to wait so can't treat people . 

Even hough early NC is pluripotential, NC cell fate is shaped by 

1. Rostrocaudal origin

2. Migration path and final location (environment) 

3. Developmental timing

A. The levels of origin of the main derivatives of the NC are indicated along the neural axis at two developmental stages: 7 somite stage (ss) in the upper left and the 28 ss (right)

B. The origin of the ganglia of the Enteric Nervous System (ENS) is inidcated in orange, that of the autonomic sympathetic chains in red and that of endocrine (adrenal meddula) in violet. 

1. Cranial (from brain region of neural tissue)

-melanocytes

- facial skeleton (cleft palate is a NC disorder)

- cranial ganglia

- connective tissue in head and neck

2. Cardiac (region of somites 1-3) 

- walls of great arteries

- aorta-pulmonary septum 

3. Vagal (region of somite 6-tail)

- melanocytes 

- dorsal root ganglia, adrenal medulla 

4. Sacral (region of somite 28 - tail) 

- enteric ganglia 

- involves bowel, bladder and reproduction function 

1. Craniofacial malformations (eg. cleft palate) often as a result of exposure o some teratogen and/or infection during first trimesters

2. Semilunar heart valve malformations (aortic and pulmonary arteries) 

3. Hirschspring's disease: failure to form enteric ganglia to innerate and maintain motility within distal large intestine and colon. 

4. Some forms of cancer (types of neuroblastoma, neurofibroma, melanoma, schwannoma, pheochromocytoma of the adrenal gland)

if have high BMP levels you have ectoderm (skin)

if have low BMP levels = neural tissues

if you have intermediate levels = neural crest cells 

- due to high BMP

- causes epidermal program 

- increase in Dlx (homeodomain transcription factor related to drosophila distal-less gene) inhibits Sox2 expression (sox2 is a marker of proliferating pluripotent neural cells of neural tube) 

 - due to low BMP (as a result of increased Noggin, etc) 

- increases Sox2 (inhibits NC genes, ex. Slug)

- increases bHLH TFs (bHLH = basic Helix-Lop-Helix TF) (Neurogenin, NeuroD)

- increase neural differentiation genes. (N-CAM, tubulin) 

due to intermediate levels of BMP

- increase neural plate border specifiers: Wnt, FGF (independent of their roles in rostrocaudal patterning) 

- increases neural crest specifiers (ex. increase in Sox9, Sox10, FoxD3)

1. Neural crest inducers --> intermediate levels of BMP and increased levels of wnt 

[pre-migration]

2. Neural plate border specifiers and neural crest cell survival --> Msx1/2, Pax3/7, zic1 [pre-migration] 

3. Neural crest transcription --> snail, foxd3, sox8/9/10 [delamination and migration] 

The first genes confer the ability to undergo an epithelial to mesenchymal transition (EMT), allowing neural crest cells to delaminate from or leave the neural tube, migrate and subsequently differentiate into diverse derivatives. These genes are activated by Wnt and other sinals, together with neural plate border markers, Zic1, Msx1/2, and Pax3/7. In addition, they repress the proliferating neural cell marker Sox2 within the neural tube. 

  CharacteristicsofNCspecifiers(transcription factors):

–  expressedinpre-andmigratoryNCCs(e.g.Snail2,Sox9,Sox10,FoxD3)

–  RepressSox2expression(markerofprolifera@ngneuraltubecells)

–  requireWntsignalforactvation

•  NCspecifiers(transcriptonfactors)regulatedownstreammediatorsofNC

migra@onanddifferentation

–  N-cadherin=proteinthataffectscell-celladhesion(cadherin=calcium

dependentadhesion)switchingN-CadherinexpressiontotypeII-cadherins,such

asCadherin7/11)allowsNCcellstomigrate(FoxD3/Sox10ac@vateCadherin-7

andWntactvatesCadherin-11

–  Posi@vefactorssuchascell-cellinterac@ons,growthfactorsalsocontributetoNC

migration

Most NC delamination signals are down-regulated in post-migratory NC cells, which results in loss of multipotential differentiation. 

•  N-CAMandN-cadherinarehomophilic

membrane-boundproteinswhichcontributeto

contactadhesionbetweenneuralcells.Inbrief,

eachmoleculecanbebothareceptorandligand

foranothermoleculeofthesametype.

•  HomophilicbindingoccursbetweenNCAM

moleculesonopposingsurfacesandNCAM

moleculesonthesamesurface.Onthesame

membrane,theyaggregatetoformcell

membraneclusters(trimersordimers)which

increasestheeffec@vebindingstrength.The

sameistrueforN-cadherin.

•  Ingeneral,foranequalnumberofhomophilic

bindingsites,N-CAMresultsinweakeradhesion

thanN-cadherin.N-cadherinhomophilicbinding

requiresCa

2+

Bothmoleculesbindtotheac@n

cytoskeletonviaβ-catenin.

•  N-CAMcanbepost-transla@onallymodifiedby

theaddi@onofpolysialicacid(PSA)which

reduceshomophilicbindingandthuscell

adhesion.PSAappearstohavearoleinsynap@c

plas@cityassociatedwithmemory.

separa@onofNCcellssepara@onfrom

neuroepithelial@ssue(ectodermand

neuraltube)andthebeginningofa

transi@onfromepithelial@ssuetoa

mesodermal@ssue

Cadherins. 

Adhesion molecules

- localized throughout lateral contact sites of neuroepithelial cells (NC precursors) 

- interact with b-catenin

-Cad6B and N-CAM expressed in premigratory cells

- Cad7 expressed in a migratory cells (subset)

cad6b is expressed in neura crest cells prior to migration.

cad7 is a type of cadherin that is not very adhesive. lost binding abilities. 

cad7 is being expressed away from each other. 

contributes to neural crest problems in development. 

can create knockout mice. this is an ex if play around with foxd3. 

ACE are control animalsthe various nerves within the forelimb of developing mouse

at the mutant, there are no neruves. sensory nerves are suppressed so you dont see stain. 

at C you can see nerves coming off of spinal cord. 

for E, arrow is pointing to enteric nervous system. at bottom, there are no staining. 

Its a complex nterplay between peripheral nerve development and formation of vertebrae from anterior and posterior sclerotome segments. 

you have to make the substrate you are growing on. If you want those cells to migrate, the substrate can't be sticky. 

extracellular matrix molecues have different adhesion capacities

the best for moving = laminin. As adult, you dont have laminin. its developmentally expressed and then goes away. Its replaced by others that are not as fluid. Part of the way to repair adult nervous system, you need laminin again. thus you need substrate for growth that allows cells to move. Once that is done, it changes the substrate that will hold the cells there and keeps it there. 

Sclerotone is the cartilaginous and bony part of vertebral column. the spine is NOT the spinal cord. they are 2 entirely different things. spinal cord is the neural tissue inside the spinal cord. 

The neural crest cells follow four different migratory paths. Exact routes vary and are dependent on rostrocaudal origin of NC cells. 

ellsthatfollowpath1become

sensoryganglia;cellsthatfollowpath2

becomesympathe@cganglia;cellsthat

followpath3willbecomeadrenal

neurosecretorycells;cellsthatfollow

path4becomepigmentedcellsand/or

entericnervous@ssue,servingthegut.

Somite:somitesarebilaterallypaired

blocksofmesoderm(segments)that

formalongtherostrocaudal(anteriorposterior)axisofthedeveloping

embryo.Somitesgiverisetoskeletal

muscle,car@lage,tendons,endothelial

cells,anddermis.

clerotome:boneandcar@lagepartofasomite(i.e.non-dermatomal,non-

myotomal)thatwillbecomethesegmentedvertebrae.Caudal(posterior)halfof

onesclerotomefuseswiththerostral(anterior)halfoftheadjacentsclerotome

toformeachvertebra.Fromini@alloca@on,sclerotomecellsmigratemedially

andsurroundthenotochord.Dorsally,thesecellsmeetthesclerotomecellsfrom

theoppositesideofthebodytoformthevertebralbody.Fromthisvertebral

body,sclerotomecellsmovedorsallyandsurroundtheneuraltube(i.e.the

developingspinalcord),formingthevertebralarchcontainingthespinalcord.

have ant and post schlerotome. in terms of migration of neural crestcells their position is important. they will follow the anterior schlerotome and never cross the post sclerotome because there are cells at post scler that push these cells away from posterior scler.

migrate at specific lanes and occur an anterior portion. 

not random nor widespread

so they are guided and directed. 

they sculpt neural crest migration patterns. 

Neuropilins(NRP-1 and NRP-2) are coreceptors (either heterodimers or 

homodimers) for secreted semaphorins(e.g. 

Sema-3A and Sema-3F). Neuropilins

associate with plexinAfor intracellular 

signaling 

Semaphorinsexcreted by posterior 

sclerotome inhibit NC cells from migrating 

within posterior sclerotome. Preferred route is 

through anterior sclerotome and myotome. 

Ephrins/Ephinhibitory signaling mechanisms 

from posterior sclerotome are also involved 

Expression of ligands & receptors influencing 

NC migration vary by NC cell type, region of 

body and developmental time 

Sclerotomesof the somiteswill form vertebrae 

fibronectin, laminin, collagen

tenascin, thrombospondin