Lecture 8: Patterning tissues: induction of the vertebrate nervous system Flashcards
Organizer, What does it do? And what are the organizers called in chick, frog, zebrafish and mouse?
- The Organiser is a conserved signalling centre that not only induces and patterns the mesoderm, but also promotes formation of the nervous system.
- Signals from the “Organiser” establish dorso-ventral and anterior- posterior axes
- Transplanting the Organiser O in a chick embryo induces a new embryonic axis with patterned mesoderm and also a nervous system
- Frog (dorsal blastopore lip/Spemann organiser), Zebrafish (Shield), Chick (Hensen’s node), Mouse (node)
Spemann and Mangold’s Organizer experiment
-Grafts of an Organiser as demonstrated by Spemann and Mangold’s experiment induce an additional body axis which contains new host derived neural tissue
-Dorsal Blastopore Lip (DBL = the
Organiser) grafted to ventral side of
unpigmented host embryo
-induces an extra embryonic axis
containing a new neural tube
Who were the discoverers of the organizer?
While Hans Spemann was awarded the Noble prize for this work, Hilde Mangold, who carried out these pioneering experiments, was killed tragically in her home when a gas cooker exploded, before the importance of her work was recognised.
Neural Induction
Neural development begins with the appearance of a morphologically defined neural plate
-diagram in notes (slide 5) showing the neural plate, primitive streak, future forebrain and future spinal cord in 19 day human embryo
Tissues with neural inducing activity
- Organiser tissue
- precursor cells of the Organiser (in chick, which includes cells of Koller’s sickle and the hypoblast itself have been shown to induce early neural genes)
- tissues derived from the organiser (axial mesoderm), e.g. notochord, (somites in the frog)
- neural plate, can induce ectoderm to form neural tissue. This is known as homeogenetic induction
Inductive Interaction
- Formation of the neural plate is an example of an inductive interaction
- “an interaction between one (inducing) tissue and another (responding) tissue as a result of which the responding tissue undergoes a change in its direction of differentiation” (John Gurdon, 1987)
- Ectoderm cells must be competent to respond to neural inducing signals
- In its simplest form COMPETENCE can be considered the expression of appropriate cell surface receptors
Gene function assays in the frog embryo
All neural inducing signals identified to date were first discovered in the amphibian embryo, which as you saw previously, is a very accessible embryo, not only for tissue transplantation experiments, but also for testing the activity of genes of interest. mRNAs can be injected into the one or two cell stage embryo and this can be grown up and the effects on neural development examined. Alternatively, this assay can be adapted to over-express a gene of interest in animal cap ectoderm. This tissue normally makes epidermis (or skin) and if it is removed early enough it can then be examined to see if it forms neural tissue in the absence of signals from the embryo… this is known as the auto-induction assay.
(diagram slide 8)
What happens if you block signalling via TGFß receptors?
The first experiment to identify a molecular component of the neural induction pathway came from studies in which signal transduction through a TGFß receptor was blocked. These experiments were carried out to assess whether the TGFb molecule activin was required for mesoderm induction.This was achieved by introducing a mutant form of the activin receptor, which lacked the intracellular domain required for receptor dimerisation and initiation of the signalling pathway inside the cell. This is known as the dominant negative approach. Embryos which did not receive this signal appeared to become empty bags of ectoderm cells which lacked all mesodermal tissue. This was the result the experimenters were looking for. However, closer examination with tissue specific markers revealed that these ectoderm cells in fact express neural markers. These experiments therefore showed one effect of blocking TGFb signalling is the formation of neural tissue.
A further piece of evidence that removal of a signal may underlie the decision to turn on neural genes came from cell dissociation experiments.
(diagram slide 9)
Cell dissociation experiment
These showed that when animal caps are removed and dissociated the cells expressed neural markers. Experiments by a number of groups (Hemmati-Brivanlou, De Robertis) have since shown that not Activin, but a related TGFb molecule BMP4 can rescue epidermal differentiation and it has been proposed that during normal development it is the blockage of BMP4 signalling that allows cells to follow a neural fate. Evidence for this comes from rescue experiments: addition of BMP4 (TGFb family member) to dissociated animal cap rescues the neurulisation effect and these cells now differentiate as epidermis. Inhibition of the BMP4 signalling pathway using dominant negative forms of receptors that bind BMPs or anti-sense BMP4 RNA leads to cells adopting a neural instead of epidermal fate. These experiments suggest that blocking signals that promote epidermal differentiation is sufficient to initiate neural development. In this sense, neural development can be considered as the default fate for ectoderm cells. This work also predicts that molecules secreted by the organiser should act to antagonise BMP signalling, and a number of such factors have now been identified…
(diagram slide 10)
Neural inducers with BMP antagonising activity
Chordin, Noggin, Follistatin and Xnr3 . These molecules are expressed in the organiser region of the frog embryo. Injection of any of these molecules at the 2-cell stage leads to the induction of early neural specific genes in the auto induction assay, that is in animal caps explants. For example, Chordin over-expression in animal caps. Chordin, Noggin and Follistatin have all been shown to bind directly to BMP4 (2 and 7) and prevent binding to its receptor(s).It is interesting that there are so many genes with similar activity. This reflects a remarkable level of functional redundancy in relation to neural induction, perhaps highlighting the fundamental importance of this process…
-(diagram slide 11)
The “default model” of neural induction
The BMP antagonising activity emanating from the Organiser, has lead to the formulation of the “DEFAULT MODEL” for neural induction.
Frog ectoderm cells will form neural tissue unless they receive signals that induce epidermal differentiation. Neural induction therefore requires signals that inhibit the epidermal inducer BMP4. BMP antagonists also pattern mesoderm in the dorso-ventral axis, Noggin , Chordin etc promoting dorsal cells types and BMP4 promoting ventral cells types… it is therefore difficult to assign BMP antagonism as a mechanism specific to neural induction, but rather, this activity is a molecular mechanism that imposes dorso-ventral pattern across the embryo.
(diagram slide 12 important!!)
Do BMP antagonists initiate neural induction
in higher vertebrates?
-Mice lacking Noggin and Chordin still form neural tissue, but other BMP antagonists may still be active.
- Fibroblast Growth Factor (FGF) signalling induces early neural genes in the chick embryo. It is required for downregulation of Bmp4 transcripts in the early epiblast, but may have additional neural inducing activities. (Add BMPR KO mice phenotype)
(diagram slide 13)
What do Factors that stimulate the MAPK pathway do and how?
-Factors that stimulate the MAPK pathway (FGF, IGF, HGF) promote neural cell fate by interfering with BMP signal transduction
- demonstrated in frog too that FGF-MAPK signalling contributes to neural induction , via MAPK inhibition of smad1 activity, an intermediary in the BMP signalling pathway that would otherwise translocate to the nucleus to activate BMP target genes. FGF, IGF and HGF can all stimulate MAPK in this assay….
(diagram slide 14, important!!)
Distinct regions of the nervous system
As we saw at the beginning of this lecture as the neural plate forms it acquires distinct characteristics in different anterior-posterior regions. This axis can be subdivided into four main regions, forebrain, midbrain, hindbrain and spinal cord. These regions also have other names, as you can see, and these are often used interchangeably! Telencephalon, diencephalon (forebrain), Mesencephalon (midbrain), Met and Myelencephalon (ant & post hindbrain), spinal column (spinal cord).
(diagram slide 16)
How are the different nervous system regions formed?
-neural mesoderm grafts
-Head and tail mesoderm induce different regions of the CNS – so neural tissue is patterned by signals from associated mesoderm
-neural anterior mesoderm grafted into early gastrula induced an extra head with eyes and forebrain
-neural posterior mesoderm grafted into early gastrula induced a trunk and tail
-graded signals from mesoderm give neural tissue a posterior identity.
-As anterior neural tissue forms first this has lead to the suggestion that initial neural inducing signals lead to the formation of anterior nervous system and that later formed mesoderm acquires the ability to induce posterior neural tissue. This observation has turned out to have a molecular correlate in the action of the BMP antagonists… which only induce neural tissue with anterior character.
(slides 17 and 18)
