Formation, Migration and Evolution of the Neural Crest

Marianne Bronner-Fraser California Institute of Technology

mbronner@caltech.edu

Amphioxus

Lamprey

Chicken stagesStage 5: 20

hours incubationStage 10: 36

hours incubationStage 16: 52

hours incubation

Neural crestarises at neural plate border during neurulation

Cell marking techniques:

1) Molecular markers

2) DiI labeling

3) Quail/chick (or duck!) chimerae

4) Image analysis

cranial

trunk

Neural crest marker gene = Slug

QuickTime™ and aVideo decompressor

are needed to see this picture.

Neural crest derived quail cells (red) in trigeminal ganglion express trkA (green)

Neural crest cell derivatives

Neural Tube

Neurons and gliaof cranial ganglia

Cartilage and bone

Connective tissue

Pigment cells

Sensory neurons/glia

Sympatho�adrenal cells

CRANIAL TRUNK

Electroporation into chick neural tube

Cell biological approaches:

1) Cell culture

2) Analysis of migratory behavior in vitro

Soluble Slit enhances neural crest migration

How do we think about neural crest formation?

Neural Crest Induced by Neural Tube/Epidermis Interactions

Wnt6 is expressed in the ectoderm

Wg or Wnt induces neural crest cells

Gain and loss of function approaches:

1) Antisense morpholino-mediated knock down

2) Over-expession by electroporation

3) Over-expression of dominant negative constructs

4) RNAi

Morpholino antisense oligos(MO) knock-down

Protein

Protein

Test the role of Pax7 in neural crest formationTest the role of Pax7 in neural crest formation

Ectopic expression on MSE55 Rho effector in neural tubecauses of epithelial cells to be mesenchymal

Control MSE55

Wnt6 is expressed in the ectoderm

inhibits Slug expressionDn Wnt

control

The Good:1) Cut/Paste Biology2) Genome Available3) Transgenesis Possible (though not simple)

The Bad:1) Genetics Difficult (some mutants but…)2) No homologous recombination3) Intermediate Visibility

Amphioxus

Lamprey

Neural crest and placodes are present only in vertebrates

Sea Lamprey: Petromyzonmarinus

Petromyzon marinus collection

Lamprey as a useful experimental model• Important taxonomic position• Manipulable embryos; egg size (1mm)• Many embryos available (≈50,000 eggs/female)• All developmental stages available• Molecular/genomic tools becoming available

– Lamprey genome project (NHGRI)– BAC library (>150kb gDNA)– Embryonic cDNA libraries (EST project)– Perturb gene function (protein knockdown) – Misexpression of foreign genes?

Lamprey developmental stages4d 6d

10d

12d

8d6d 8d

10d

12d

DiI labeling of neural crest in Petromyzon

Neural crest migration comparison

Knocking down PmSoxE1 protein using morpholino

antisense oligos (MO)

Protein

Protein

The Good:1) DiI Labeling Possible2) Genome Available Soon3) Morpholinos Work

The Bad:1) Genetics Out of the Question2) Opaque Embryo--Imaging is Out

The Ugly:1) Breed One Month/Year 2) Embryos Hard to Get

Neural Crest Induction Via Cell Interactions at Neural Plate Border

Steps:1. Induction Signals2. Neural Plate Border Specification3. Neural Crest Specification

Goal:Dissect Gene Regulatory Network

Putative Vertebrate Gene Regulatory Network

Putative Vertebrate Gene Regulatory Network

Putative Vertebrate Gene Regulatory Network

SummarySteps in Network:

• INDUCTION: Cell interactions mediated by Wnts and other signals (BMP, FGF) induceneural crest

• SPECIFICATION: Pax7 required for neural crest--regulates Slug, Sox9, and Sox 10 FoxD3

• DOWNSTREAM TARGET: Slug represses Precrest

Amphioxus

Lamprey

Question:

What elements of gene network are conserved?What genetic changes drove neural crest evolution?

Experimental Approach:

Compare “neural crest gene” expression, function &regulation in amphioxus, agnathan & gnathostomes

Amphioxus has a single AP-2 gene

expressed in ectoderm

Early lamprey AP-2 is expressed in ectodermLater lamprey AP-2 is expressed in dorsal

neural tubeand neural crest

Lamprey AP-2

Vertebrate neural crest markers arenot co-expressed in Amphioxus

Neural Plate BorderVertebrate

Evolution of Neural Crest Likely Involved Co-option of Existing Genes to New Neural Crest SpecifierFunction

Neural Plate Border

AmphiSnail

?

Amphioxus

Gene expression implies cooption by vertebrateneural crest

New gene deployment at neural plate border

New function of “old genes”

What could this mean?

Martin Garcia-Castro David McCauleyMartin Basch Lisa TaneyhillDan Meulemans Ed ColesCelia Shiau

Scott Fraser