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.
History of Neural Induction Hypothesis 1.Spemann Organizer ( ) 2.Default Model ( ) 3.Neural Induction in Chick and Mouse (2001-)
Induction of Embryonic Primordia by Implantation of Organizers from a Different Species Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech. 100, , 1924
Development of Xenopus embryo Early gastrula: Dorsal blastopore lip
Fate map of the Xenopus gastrula (Hemmati-Brivanlou & Melton, 1997)
Classical Transplantation Experiment by Spemann and Mangold Dorsal blastopore lip The donor tissues could recruit the host cells to become the secondary neural tube. (Hemmati-Brivanlou & Melton, 1997)
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. “Spemann Organizer” Epidermis: “Default” fate for gastrula ectoderm Neural specification: needs a positive signal from neighboring cells (Neural Induction). “Default”: Cell autonomous.
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.
Animal Cap Assay A V In vitro culture Markers expression (RT-PCR) : Mesoderm Neural ectoderm Epidermis Endoderm Animal cap Blastula (stage 8) In early 1980, TGF- and FGF family members have been found to have the mesodermal and neural induction activities. Questions: Posterior neural ectoderm? No mesoderm induction?
Inconsistency with Neural Induction Hypothesis A V In vitro culture Epidermis Dissociated into signal cell, 4-5hr Reaggregation and culture Neural Tissue Animal cap Blastula (stage 8) (Grunz & Tacke, 1989; Godsave & Slack, 1991) 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.
Ali Hemmati-Brivanlou & Douglas Melton Nature, 1992, 359, 1XAR1 In vitro culture Neural Tissue 2-cell stage Blastula Truncated activin receptor induces neural tisse XAR1 1XAR1 Questions raised: 1.Activin is a mesoderm inducer, not a neural inducer. 2.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. Organizer MesodermNeural tissue
Stern, Development, 2005 Experiments in Xenopus that support the default model
Stern, Development, 2005 The “default model” in Xenopus Question: How do these BMP inhibitors antagonize BMPs’ function?
By Eddy De Robertis Group in UCLA
Chordin protein is secreted by cultured cells and by Xenopus organizer tissues Chordin is a 120 kDa secreted protein. VMZDMZ
How could Chordin antagonize BMPs’ signaling? 1.Interfere with BMP maturation or secretion; 2.Act via a parallel pathway to that of the BMP-receptor interaction; 3.Bind to the BMP receptor; 4.Bind directly to mature BMPs.
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)
Chordin inhibits BMP-4 binding to its receptor (100X)
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 TGF 1.
Cross-linking analysis confirms the direct bind of Chordin and BMP4
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.
Neural induction and mesoderm dorsalization by Chordin protein and was inhibited by BMP-4 Neural induction (without mesoderm induction) Mesoderm dorsalization (ventral marginal zone)
Molecular mechanism of Chordin antagonizes BMP4 signaling
By Richard Harland Group at UC Berkeley
Default Model in Chick and Mouse Early Development Questions unsolved: 1.In HNF3 KO mice, there is no node, but the embryos have the neural tissues (Node = Organizer in Xenopus). 2.Neural induction is initiated before gastulation. 3.BMP antagonists are not required for neural induction.
An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo Wilson et al., Curr. Biol., 2000, 10,
Neural markers Specification of cells of the stage XII chick embryo Epidermal In stage XII chick embryo, medial epiblast differentiate into neural cells, while lateral epiblast acquire the epidermal character.
BMP4 BMP7 FGF3 S17 FGFR2b S17 FGF signaling and BMP expression in stage XII epiblast cells MedialLateral Bef. Cul. Cul. 40h 40h+ SU 40h+ SU+Nog Bef. Cul. Cul. 40h Acquisition of neural character is accompanied by repression of BMP4 and BMP7 expression.
BMP4 induces epidermal character in prospective neural cells in stage XII BMP4 1.3nM + Medial
The FGFR tyrosine kinase inhibitor SU5402 inhibits the acquisition of neural character Medial + SU5402 Medial + SU5402 +Noggin Medial +SU5402 +IB/Fc +IA/Fc Noggin: BMP binding protein IB/Fc & IA/Fc: soluble dominant negative BMP receptors.
The status of Wnt signaling regulates neural and epidermal fates in the chick embryo Wilson et al., Nature, 2001, 411,
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.
Neural MarkersEpidermal Markers Regulation of neural and epidermal fate in medial epiblast
Neural Markers Epidermal Markers Regulation of neural and epidermal fate in lateral epiblast
Summary of Experiments
FGF, WNT and BMP play important roles in neuralization of amniote embryos (humans, rodents and birds) Wilson et al., Nature Neurosci., 2001 An unifying mechanism of “neural induction” ?
Stage XI-XII Stage XIII-2 Stage End of Stage 4 ??? Neural induction in chick embryos ---Embryologist’s view
Neural induction in chick embryos ---Genetic cascade
Models of neural induction Xenopus Chick
Establishment of A-P axis in neural plate Two-inducer model: Anterior and posterior neural inducers Two-step model: Nieuwkoop's activation–transformation model
Early mouse development Preimplantation
Postimplantation Early mouse development
Distal VE to AVE Cell movements in early mouse embryos Epiblast to posterior Extraembryonic ectoderm to posterior epiblast Epiblast to posterior
Otx2 VEcis-lacZ 5.5 dpc 5.75 dpc 6.25 dpc 6.0 dpc Otx2 KI-lacZ Movement of Otx2-positive cells from DVE to AVE DVE: distal visceral endoderm; AVE: anterior visceral endoderm