8 Major Steps in Neural Differentiation Competence: Cells have the ability to become neural precursors if they are exposed to the right combination of signals.Specification: Cells have received the signals to become neural precursor cells but will still respond to signals that repress a neural character.Commitment: Cells have received the signals to become neural precursor cells and will progress to become neurons even in the presence of signals that repress a neural character.Differentiation: Neural precursor cells exit the cell cycle to become post-mitotic neurons.
9 History of Neural Induction Hypothesis Spemann Organizer ( )Default Model ( )Neural Induction in Chick and Mouse (2001-)
10 Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech. Induction of Embryonic Primordia by Implantation of Organizers from a Different SpeciesHans Spemann and Hilde MangoldArch. Mikr. Anat. Entw. Mech.100, , 1924
11 Development of Xenopus embryo Early gastrula: Dorsal blastopore lip
12 Fate map of the Xenopus gastrula (Hemmati-Brivanlou & Melton, 1997)
13 Classical Transplantation Experiment by Spemann and Mangold The donor tissues could recruit the host cells to become the secondary neural tube.Dorsal blastopore lip(Hemmati-Brivanlou & Melton, 1997)
14 “Spemann Organizer”Spemann named the dorsal blastopore lip the “organizer”, and proposed that in normal development this region induces and organizes a correctly patterned nervous system in neighboring dorsal ectoderm. In the absence of this influence, as on the ventral side, the ectoderm differentiates as epidermis.Epidermis: “Default” fate for gastrula ectodermNeural specification: needs a positive signal from neighboring cells (Neural Induction).“Default”: Cell autonomous.
16 This hypothesis dominated the developmental biology field for several decades. A considerable effort over several decades failed to identify the gene products responsible for neural induction in the embryo.
17 Markers expression (RT-PCR) : Animal Cap AssayAVIn vitro cultureMarkers expression (RT-PCR) :MesodermNeural ectodermEpidermisEndodermAnimal capBlastula(stage 8)In early 1980, TGF-b and FGF family members have been found to have the mesodermal and neural induction activities.Questions: Posterior neural ectoderm?No mesoderm induction?
18 Inconsistency with Neural Induction Hypothesis (Grunz & Tacke, 1989; Godsave & Slack, 1991)AVIn vitro cultureEpidermisDissociated into signal cell, 4-5hrReaggregationand cultureNeural TissueAnimal capBlastula(stage 8)The idea of a positive signal involved in neural induction so dominated thinking in the field that the significance of results inconsistent with this idea were not widely appreciated.
19 Truncated activin receptor induces neural tisse Ali Hemmati-Brivanlou & Douglas MeltonNature, 1992, 359,D1XAR1In vitro cultureNeural Tissue2-cell stageBlastulaQuestions raised:Activin is a mesoderm inducer, not a neural inducer.The block in activin signal transduction autoinduces cells of the animal cap to switch to a neuronal fate in the absence of any detectable mesoderm.XAR1D1XAR1???OrganizerMesodermNeural tissue
20 Experiments in Xenopus that support the default model Stern, Development, 2005
25 Chordin protein is secreted by cultured cells and by Xenopus organizer tissues VMZDMZChordin is a 120 kDa secreted protein.
26 How could Chordin antagonize BMPs’ signaling? Interfere with BMP maturation or secretion;Act via a parallel pathway to that of the BMP-receptor interaction;Bind to the BMP receptor;Bind directly to mature BMPs.
27 Chordin protein inhibits the osteogenic activity of mature BMP proteins AP activity can also be induced by RA, but this activity could not be inhibited by Chordin. (BMP specific inhibition)
28 Chordin inhibits BMP-4 binding to its receptor (100X)(100X)
29 Chordin binds to BMPs but not to Activin Chd-BMP4 interaction could not compete by other GFs.Chd-BMP4 interaction could compete by BMP2, but not by activin and TGFb1.
30 Cross-linking analysis confirms the direct bind of Chordin and BMP4
31 Chordin binds to BMP4 with high affinity Chd could bind to BMP4 with the same affinity as BMP4 binds to its receptor.Chd could bind to BMP4 homodimer and BMP4/BMP7 heterodimer with same affinity.
32 Neural induction and mesoderm dorsalization by Chordin protein and was inhibited by BMP-4 (without mesoderm induction)Mesoderm dorsalization (ventral marginal zone)
33 Molecular mechanism of Chordin antagonizes BMP4 signaling
35 Default Model in Chick and Mouse Early Development Questions unsolved:In HNF3b KO mice, there is no node, but the embryos have the neural tissues (Node = Organizer in Xenopus).Neural induction is initiated before gastulation.BMP antagonists are not required for neural induction.
36 An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo Wilson et al., Curr. Biol.,2000, 10,
37 Specification of cells of the stage XII chick embryo Neural markersEpidermalIn stage XII chick embryo, medial epiblast differentiate into neural cells, while lateral epiblast acquire the epidermal character.
38 FGF signaling and BMP expression in stage XII epiblast cells MedialLateralBef. Cul.Cul. 40h40h+ SU40h+ SU+NogBef. Cul.Cul. 40hBMP4BMP7FGF3S17FGFR2bAcquisition of neural character is accompanied by repression of BMP4 and BMP7 expression.S17
39 BMP4 induces epidermal character in prospective neural cells in stage XII BMP4 1.3nM+ Medial
40 The FGFR tyrosine kinase inhibitor SU5402 inhibits the acquisition of neural character Medial+ SU5402Medial+ SU5402 +NogginMedial +SU5402+IB/Fc +IA/FcNoggin: BMP binding proteinIB/Fc & IA/Fc: soluble dominant negative BMP receptors.
41 The status of Wnt signaling regulates neural and epidermal fates in the chick embryo Nature, 2001, 411,Wilson et al.,
42 Wnts, FGF3 and BMP4 expression in medial and lateral epiblast of chick embryo M: Medial epiblast; L: Lateral epiblast.mFrz8CRD-IgG: Soluble Frizzle 8, Wnt receptor inhibitor.
43 Regulation of neural and epidermal fate in medial epiblast Neural MarkersEpidermal Markers
44 Regulation of neural and epidermal fate in lateral epiblast Neural MarkersEpidermal Markers