Neurogenesis and NeuronalMigration

Paul Garrity

March 1, 2004

7.68J/9.013J

Development of corticallayers

• Cortex starts outas monolayerepithelium

• Nuclei/cells moveup and downaccording to theircell cycle phase

Development of corticallayers

• Neurogenesisinitiates:– Some cells begin to

leave cell cycle -- rise• Form preplate

– Cajal-Retzius cells– Subplate cells

– Many cells continue todivide

• Ventricular zone (VZ)

– Axons enter cortex:Intermediate Zone (IZ)(bidirectional cortex/thalamusconnections)

Subplate cells -

Cajal-Retzius cells -

Development of corticallayers

• Newly generatedneurons migratethrough subplate

• Stop beneathCajal-Retzius cells

• Form cortical plate

Development of corticallayers

• Cortical Platedifferentiatesto formcortical layers

Development of corticallayers

• As corticalplate forms

• Subpopulationof proliferatingcells formsabove VZ :

• SubventricularZone (SVG)

Subventricular Zone

• Secondary zone ofneurogenesis

• Proliferate through post-natal period– Generate multiple cell

types:• Glia

• Neurons

• Include cells that migrate toolfactory bulb

Are cortical layersgenerated in any temporal

sequence?• Birthdating

analysis• Inject mother with

tritiated thymidine• Label dividing cells• Diluted with

subsequentdivisions

Birthdating of cortical layersin rodents

• Inject at multipletime points

• Detect usingautoradigraphy

• Answer: Corticallayers generated“inside-out”

Birthdating in monkeycerebral cortex

Primates: Cortical layers

also generated“inside-out”

Radial migration

• Post-mitoticneurons migrateaway fromventricular zonetoward brainsurface

Pattern of migration

• Newlygenerated cellsmigratebeyond earliercells

Neuron growing along glialcell in culture

Migration along radial glialcells

• Radial glial cellsspan thedevelopingcortex

• Neurons appearto migrate inclose contactalong them

Neuronal migration

How is neuronal migrationregulated?

• Molecularpathwayscontrollingneuronalmigrationidentifiedthroughhuman andmousemutants:

The reeler mutant mouse

• Birthdatinganalysis ofreeler mutant:

The reeler mutant mouse

• Birthdatinganalysis ofreeler mutant:

• Timing oflayerproduction isinverted

Anatomy of developingcortex in reeler

wild type reeler

Molecular identification ofReelin

• Secreted protein

• Produced by Cajal-Retziuscells

wild type reeler

Reelin

Model for Reelin function

• Signal promotingmigration alongand/or detachmentfrom radial glial cell

Reelin

The Reelin pathway

• Other mutant mice found with samephenotype: eg., Dab1

Dab1

• Cytoplasmic adaptor protein

• Binds to receptors

• Binds to cytoplasmic protein kinases

Receptors for Reelin

• Animals double mutantfor ApoER2/VLDLRresemble reeler

• Well-known lipoproteinreceptors

• Expressed in migratingneurons

• Bind ReelinReelin also binds integrins --

co-receptor?

Reelin signaling pathway

ApoER2/VLDLRbind Dab1!

-- in addition:mutants in P35and cdk5 (whichfunctiontogether askinase complex)have similarphenotypes toreeler

Disorders of neuronalmigration in human disease

enlarged ventriclesreduced white matter

decreased folding

cerebellum

Regulators of migrationfound as human disease

genes

Lissencephaly(smooth brain)

Cobblestone cortex

Corticalheterotopia[sideview]

Cortical layering in patients

Schematic of how layeringdefects may be generated

Normal Classical Lissencephaly Cobblestone cortex Cortical Heterotopia

Cobblestone cortex

• Abnormal basallamina/extracellular matrix– Fukutin: glycoprotein/glycolipid

modifying enzyme

– Muscle-eye-brain (MEB) diseaseprotein: protein glycosylatingenzyme

– May disrupt basal laminasurrounding brain

Lissencephaly genes:microtubule regulators involved

in nuclear migration• Genes that interact with microtubules:

– Lis1 (homolog of NudF -- required for nuclearmigration in Asperigillus nidulans )

• Interacts with microtubule organizer (centrosome)• Interacts with Dynein (microtubule motor protein) --

multiple roles including nuclear movement

– DCX (microtubule binding protein)

X-linked periventricularheterotopia

• Mutant in Filamin– Actin-associated protein

– Associates with multiple regulators ofactin cytoskeleton

• Both actin and microtubulecytoskeletons important in migration

Tangential migration incortex

• Embryological and labellingexperiments demonstratedthat not all cortical cells arisefrom radial migration

• Lose GABA-ergicinterneurons in mutant micewith disrupted LGE and MGEdevelopment

• GABA-ergic interneuronsmigrate in from region ofbasal telencephalon (medialganlionic eminence, MGE)

Molecular mechanisms oftangential migration

• Differs from radial migration:– Does not require reelin, dab or cdk5

Regulators of tangentialmigration

• Semaphorins: family of guidancecues: attract and repel cells andprocesses– Sema 3: secreted signal

Regulators of tangentialmigration

• Semaphorin 3 receptors:– Neuropilin (ligand-binding subunit)

– Plexin (trans-MB signal transducer)

– L1 (modulator)

Semaphorin signaling intangential migration

• Neuropilin (receptor) expressed onmigrating cells

• Semaphorin 3 expressed on pathway• Examined effect of disrupting

Neuropilin signaling via:– Nrp2 knock-out mouse– Nrp1 dominant-negative

• How to make a dominant-negative receptor?

Dominant-negativeneuropilin

• Truncation of cytoplasmic domain– No effect

Neuropilin dominant-negative receptor

• Neuropilin functions:– Bind Sema 3

– Initiate signal transduction

Dominant-negativeneuropilin

• Truncation of cytoplasmic domain– No effect

• Truncation in extracellular domain-Dominant-negative

-Still binds Sema3

-Signaling fails

Dominant-negativeneuropilin

• Truncation of cytoplasmic domain– No effect

• Truncation in extracellular domain-Dominant-negative

-Still binds Sema3

-Signaling fails x

Neuropilin signaling regulatestangential migration

Dorsal/Ventral Axispatterning

• Structures along DVaxis of Neural Tube– Roof plate (R)

– Floor plate (F)

– Notochord (N)

– Neural crest (NC)

– Paraxialmesoderm/somites (S)

Neural Crest Cells

Generation of appropriatenumbers of cells

• A) Non-self-renewingprogenitor:generates twodifferentiating cells

• B) Self-renewing:generates at leastone cell same asparent

Nervous system progenitors

• Nervous systemsundergo enormousexpansion in cellnumber duringdevelopment

• Relies on cells that canself-renew: stem cells

Stem cell divisions• Symmetric

division:– Generates two

stem cells

•Asymmetricdivision:

–Regenerates stemcell and produces anovel cell

Stem cells in thehematopoietic system

• Plutipotent stem cellscan generate stemcells withprogressivelyrestricted potentialfates

• Restriction canproceed in more thanone step as generateincreasinglycommitted progenitors

Neural stem cells

• Key properties:

• Multipotent -- generate multipledifferent types of progeny

• Self-renewing

Sample genealogy ofcortical neuronal stem cell

• Self-renewing

• Undergo symmetric(diamond, circle) andasymmetric (*)divisions

• Multipotent: generatesneurons (N) and glia (_)

Stem cell divisions• Symmetric

division:– Generates two

stem cells

•Asymmetricdivision:

–Regenerates stemcell and produces anovel cell

Shifts in fraction of patternof stem cell division with

time

Radial glial cells:

• Classic view:• Radial glial cells act as substrates for

neural migration• A distinct population of cells generates

neurons

Radial glial cells: (c. 2001)

• Radial glial cells are mitotically active• What do they produce?

– Infect radial glia with GFP retrovirus– Identify single, labelled radial glia cells at 24h– Wait 2 more days ( forms a clone of cells )

Radial glial cells: more thanjust substrate for migration

• What do labelled radialglial cells produce?– See labelled:

• mitotically active radial glia --divide in VZ

• post-mitotic neurons

– Post-mitotic neuronsmigrate along clonallyrelated radial glial cells --

Fishell and Kriegstein (2003) Current Opinion in Neurobiology 13:34

Current models for RadialGlial Cell asymmetric

division

Fishell and Kriegstein (2003) Current Opinion in Neurobiology 13:34

Current models for RadialGlial Cell asymmetric

division• Current evidence

suggests thatboth“translocation”and “migration”are used

Fishell and Kriegstein (2003) Current Opinion in Neurobiology 13:34