Neural induction

Neural induction

1. 1. BACKGROUND (Xenopus)

   • a.Development
     • Gastrulation - formation of three germ layers
       • Begins as a small involution of smooth surface opposite the sperm entry site
         • This is called the dorsal lip of the blastopore OR involuting marginal zone (IMZ
           • It forms the blastopore
             • The mesoderm is the first layer to move inwards and underlies the prospective neurogenic region
               • Involution occurs via a coordinated contraction of actin and microfilaments
                 • Neural induction transforms this neurogenic region to form the neural plate which forms neurons and glia of the brain and spinal cord from the neural tube
                   • The PNS forms from the neural crest
                 • The mesoderm condenses to form the notochord
       • Details of the movement varies between species but all form the three germ laters
   • b. When does the ectoderm acquire neural fate
     • Initially take the neural ectoderm regions fated to make the nervous system as what's known as an animal cap
       • REMOVING ECTODERMAL TISSUE PREGASTRULATION
         • Produces epidermis
       • REMOVING ECTODERMAL TISSUE POSTGASTRULATION
         • Produces neural tissue
       • What is the difference, apart from time?
         • The tissue arrangements of gastrulation is critical (mesoderm MUST go under the epidermal layer during gastrulation)
           • Simplest explanation: mesoderm tissue is telling the overlying tissue to become neural tissue
   • c. Speeman-Mangold Organiser
     • i. Experiment
       • Taking the dorsal lip of an early gastrula donor and transplanting it into another region of the host into the opposite region
         • This forms a second axis - the tissue is competent to start gastrulation despite being at a different ectopic region
           • Therefore, the DLB can induce neural tissue to form where it doesn't normally AND organizes the entire body axis
       • This organizer activity is conserved across species
         • Chick Hensen's node (bird DLP) transplant
           • Transplanting duck embryo tissue into a chick embryo forms a primary nervous system (showing foreign tissue can still induce nervous system formation)
           • Adding Hensen's node into amphibian and mammal species ALSO works
         • Zebrafish 'shield' transplant
           • Transplantation within different species also forms a second nervous system
     • ii. What it tells us?
       • 1. THE MESODERM INDUCES THE OVERLYING ECTODERM TO DEVELOP AS A NERVOUS SYSTEM
       • 2. ORGANISER ACTIVITY IS CONSERVED ACROSS SPECIES
       • 3. ORGANISER DOES NOT LOSE ITS NEURALIZING EFFICACY WHEN HEATED, FROZEN OR TREATED WITH ALCOHOL

2. 2. 6 EXPERIMENTS EXPLORING THE NEURALISING TISSUE, WHAT THE SUBSTANCES ARE AND HOW THEY WORK

   • a. Isolating the inducer
     • i. What we know already about fates in Xenopus:
       • Animal cap: produces neural and ectoderm tissue
       • Vegetal cap: produces endoderm (gut) tissue
       • Mesoderm tat the equator produces muscle, blood, bone etc.
     • ii. Experiment:
       • Taking an animal cap assay and putting it to one side leads to the mesoderm and neuroectoderm failing to develop
         • Removing the area fated to be mesoderm and placing the V + A caps together reproduces a mesoderm
           • Therefore, diffusible signals from the V pole induces mesoderm formation
             • Addition of factors to ONLY an A cap leads to mesoderm and neural formation
   • b. Isolating the inducer
     • Testing an A cap with mesoderm inducers leads to mesoderm formation, which signals the A pole to become neural tissue
       • BUT, we want to form neural tissue without the mesoderm layer
         • We therefore must discover the neural tissue inducers/exogenous factors rather than direct mesoderm inducers...
           • THEREFORE, we need to isolate the factor that directly induces neural fate… directly upregulates neural gene expression (markers) without the induction of mesoderm specific markers
   • c. Isolation of Noggin - the neural tissue inducer produced in the DLP
     • i. Experiment
       • Experiments can be done to hyperdorsalise or hyperventralise an embryo
         • Using UV on a Xenopus embryo causes it to become ventralised
           • As UV intervenes with the formation of microtubules causing no dorsal structures to form
             • Transplanting the DLP into the UV embryo can rescue it and restore its axis
         • Using lithium (Li) dorsalises the embryo
         • Injecting a hyperdorsalised embryo with mRNA from a hyperventralised embryo leads to rescue
           • Therefore, to identify the neural inducer, Richard Harland's group prepared cDNA from the organizer region in ventralised embryos
             • This is done via Sib selection: taking populations of cDNA and seeing if they have an effect. If not they're forgotten, if they do then they're divided into smaller populations
               • This identified Noggin! (as it induced a head structure)
                 • Recombinant Noggin protein added to animal caps – and/or ventralised embryos induces neural genes directly and rescues NS
                   • Expressed in the DLB
   • d. Chordin expression
     • Discovered in a similar way to Noggin
     • It is expressed in the dorsal lip of the blastoderm
     • Neural induction by Chordin is direct and not through a transient mesoderm phase
       • Animal caps prepared from chordin mRNA-injected embryos do not form mesoderm but neural cell types directly
         • HOWEVER, NO ONE KNEW HOW CHORDIN OR NOGGIN WORKED
   • f. Follistatin-Activin interaction
     • A screen for mesoderm inducers isolated the signaling molecule Activin (Melton) a TGFβ member
     • i. Mechanism of Activin function
       • Is a diffusible molecule
       • Its receptor is membrane bound
       • Generated truncated Activin receptors to block the Activin signal
         • Then inserted mRNA encoding them into animal cap assays
           • This resulted in dimerization of truncated receptors with wt receptors
             • The product = no mesoderm or epidermis (as a normal A cap would produce) BUT NEURAL TISSUE!
               • THEREFORE - ACTIVIN INHIBITS NEURAL INDUCTION, INHIBITION OF THIS INHIBITOR RESULTS IN DEFAUL STATE NEURAL A.K.A DEFAUL HYPOTHESIS
     • ii. Mechanism of Follistatin function
       • Follistatin binds with Activin and prevents it from having its inhibitory effect (inhibits the inhibitor) and leads to neural infuction
   • e. Cell dissociation experiments
     • Occurred before activin discovery
     • Dissociation of an animal cap results in neurons forming as the inhibitory substance (activin) cannot function
       • Adding BMP4 (a TGFBeta member) resulted in the dissociated animal cap forming epidermis
         • It also originates in the DLB + axial mesoderm
         • THEREFORE, FOLLISTATIN IS THE THIRD NEURAL INDUCER AS IT IS:
           • i. An activin antagonist
           • ii. Expressed in the DLB and axial mesoderm
           • iii. mRNA can induce a second axis
           • iv. Protein can induce neural tissue from intact animal cap

3. How is neural tissue induced? What is the source of induction?

   • 4. THE DEFAULT HYPOTHESIS
     • a. The hypothesis
       • Neural induction (Noggin, Chordin and Follistatin) by inhibiting the inhibitor
         • Whereas the classic idea = the ectoderm was induce to become neural tissue because of active induction from the mesoderm
           • We now know that normally activin like proteins of the TFGβ family prevent neural induction by binding on their receptor and preventing neural tissue forming
             • BUT substances such as Noggin, Chordin and Follistatin bnd onto these TFGβ molecules and prevent them from binding to their receptor to allow neural tissue to form
     • b. How do neural inducers work?
       • As the involuting mesoderm tissue does underneath the overlying neural plate, it secretes BMP antagonists inclding Follistating, Chordin and Noggin diffusing molecules to inhibit the inhibitor
         • i. Chordin
           • Binds with BMP4 with high affinity
         • ii. Noggin
           • Binds BMP4 with even greater affinity
         • iii. Follistatin
           • Binds with BMP7 (and Activin)
         • They block the anti-neuralising effects of the BMP signalling by sequestering free BMPs around the midling
     • c. Position dependent model of neural induction
       • If the BNP inhibits wnt signalling then the brain forms
         • BUT if wnt is allowed to activate tissue you can observe the development of the nervous system
     • d. Examples

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