What type of molecule is NF - protocadherin?
Homophilic cell adhesion molecule
Where is NF-protocadherin expressed?
Expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis (african clawed frog)
What is the result of targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium?
Unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract
Semaphorin 3A is said to lie adjacent to this caudal turn in the mid-optic track, what is its action in vitro and in vivo?
In vitro - stimulates rapid, protein synthesis–dependent increases in growth cone NFPC and its cofactor, TAF1
In vivo - growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function.
What is the pathway of the retinal ganglion cells?
Retinal ganglion cells (RGCs) send axons out of the retina, along the optic nerve, across the chiasm and up the ventral optic tract (VOT). In the mid-optic tract (MOT), they change direction, turn- ing caudally toward the tectum
retina - optic nerve - chiasm - ventral optic tract - mid optic tract - tectum
What prevents the retinal axons from entering the telencephalon or extending too far dorsally?
Repellent cues Slit1, Slit2 and Sema3A
They demarcate the rostral and dorsal boundaries of the optic tract
What is the result of Sem3A and Slit 1 knockout?
Axon stalling at the caudal turn in the MOT suggesting that such repellants may also somehow promote the next leg of the journey to the tectum.
What does cadherin stand for?
Calcium dependant adhesion
What is the structure of cadherins?
Each cadherin has a small C-terminal cytoplasmic component, a transmembrane component, and the remaining bulk of the protein is extra-cellular (outside the cell).
How do cadherins allow cell-cell adhesion?
Cadherins form homodimers
the conformational change of cis-dimers to trans-dimers allows cadherins to create cell-cell adhesion with cadherins from other cells
Which molecules link the cadherins to the actin filaments within the cell?
Catenins (alpha, beta and gamma)
Vinculin
What is the role of cadherins in the nervous system?
Critical in establishing neuronal circuitry
Particularly in axon initiation and synapse formation
What is an ortholog?
By definition, orthologs are genes that are related by vertical descent from a common ancestor and encode proteins with the same function in different species
NFPC is the ortholog of which gene?
NF-protocadherin (NFPC) belongs to the non-clustered protocadherin subgroup of this superfamily and is the Xenopus ortholog of Pcdh7.
What is the structure of NFPC?
It consists of seven cadherin-like ecto- domains, a single transmembrane domain and a C-terminal intracellular domain that binds its cytosolic partner, TAF1 (also known as TAF-I and SET).
What is the role of NFPC during embryogenesis?
During embryogenesis, NFPC regulates ectodermal cell sorting and neural tube closure through homophilic adhesive interactions mediated by TAF1
NFPC is also strongly expressed in growing RGC axons and in the optic tract neuroepithelium during the development of the Xenopus retinotectal projection
What evidence suggests that NFPC guides growing axons through the caudal turn to the tectum?
The expression of NFPC in the neuroepithelium demarcates the mid-to-dorsal optic tract and precedes retinal axon entry into this region
What is the significance of NFPC mRNA being present in the RGC axons?
Adhesive interactions can be translationally regulated by local cues
Why is semaphorin 3A and slit 1 though to be linked to NFPC and TAF1?
Targeted disruption of NFPC or TAF1 in either the axons or the neuroepithelial substrate leads to specific pathfinding defects in the MOT. The defects were similar to those seen with the combined knockdown of Sema3A and Slit1.
'axon stalling at the caudal turn in the MOT'
What is the relationship between semaphorin 3 and NFPC?
Exploring this potential link, we found that Sema3A, but not Slit, triggered a rise in NFPC in growth cones through local protein synthesis
What type of signalling is responsible for the NFPC translation-reporter activity at the caudal turn?
Depends partly on the semaphorin - neurolipin signalling
Together, the results show that NFPC-NFPC adhesion helps to guide axons at the caudal turn and suggest that cell adhesion can be spatially modulated by regionally expressed cues by means of a translation-linked mechanism.
In a previous study, we found that expression of dominant nega- tive NFPC and TAF1 constructs (NF∆E and TAF1∆N, respectively) during early retinal differentiation inhibited axonogenesis in RGCs
How can you examine the role of NFPC and TAF1 in axonogenesis if negative constructs results in inhibited axonogenesis?
Inhibition of function has to be after axon initiation.
We wait until the RGC axons have reached the optic chiasm
After this, by using electroporation, NF∆E-myc and TAF1∆N-myc are delivered into the retina
What is the appropriate control this experiment - looking at the pathways of axons which have negative constructs that affect their development
Axons electroporated with an myc vector or a vector containing GFP only.
What is the effect of replacing NFPC and TAF1 with NF∆E-myc or TAF1∆N-myc?
Axons stopped short of the tectum and exhibited abnormally high numbers of growth cones that appeared to be stalled in the optic tract
They were significantly retarded in their navigation in comparison to transfected controls
What is another method that was used to knock out NFPC and TAF1?
We used antisense morpholino oligonucleotides (morpholinos) as a further approach to knock down NFPC and TAF1 function
How would you evaluate efficient knockdown by the morpholinos?
Western blot analysis
What is the purpose of a western blot?
Detect specific proteins in a sample of tissue homogenate or extract.
What are the stages of a western blot?
In brief, the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognises and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognises and binds to the primary antibody. The secondary antibody is visualised through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.
