Viva Paper

Question 1

What type of molecule is NF - protocadherin?

Answer 1

Homophilic cell adhesion molecule

Question 2

Where is NF-protocadherin expressed?

Answer 2

Expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis (african clawed frog)

Question 3

What is the result of targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium?

Answer 3

Unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract

Question 4

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?

Answer 4

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.

Question 5

What is the pathway of the retinal ganglion cells?

Answer 5

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

Question 6

What prevents the retinal axons from entering the telencephalon or extending too far dorsally?

Answer 6

Repellent cues Slit1, Slit2 and Sema3A

They demarcate the rostral and dorsal boundaries of the optic tract

Question 7

What is the result of Sem3A and Slit 1 knockout?

Answer 7

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.

Question 8

What does cadherin stand for?

Answer 8

Calcium dependant adhesion

Question 9

What is the structure of cadherins?

Answer 9

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).

Question 10

How do cadherins allow cell-cell adhesion?

Answer 10

Cadherins form homodimers

the conformational change of cis-dimers to trans-dimers allows cadherins to create cell-cell adhesion with cadherins from other cells

Question 11

Which molecules link the cadherins to the actin filaments within the cell?

Answer 11

Catenins (alpha, beta and gamma)

Vinculin

Question 12

What is the role of cadherins in the nervous system?

Answer 12

Critical in establishing neuronal circuitry

Particularly in axon initiation and synapse formation

Question 13

What is an ortholog?

Answer 13

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

Question 14

NFPC is the ortholog of which gene?

Answer 14

NF-protocadherin (NFPC) belongs to the non-clustered protocadherin subgroup of this superfamily and is the Xenopus ortholog of Pcdh7.

Question 15

What is the structure of NFPC?

Answer 15

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).

Question 16

What is the role of NFPC during embryogenesis?

Answer 16

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

Question 17

What evidence suggests that NFPC guides growing axons through the caudal turn to the tectum?

Answer 17

The expression of NFPC in the neuroepithelium demarcates the mid-to-dorsal optic tract and precedes retinal axon entry into this region

Question 18

What is the significance of NFPC mRNA being present in the RGC axons?

Answer 18

Adhesive interactions can be translationally regulated by local cues

Question 19

Why is semaphorin 3A and slit 1 though to be linked to NFPC and TAF1?

Answer 19

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’

Question 20

What is the relationship between semaphorin 3 and NFPC?

Answer 20

Exploring this potential link, we found that Sema3A, but not Slit, triggered a rise in NFPC in growth cones through local protein synthesis

Question 21

What type of signalling is responsible for the NFPC translation-reporter activity at the caudal turn?

Answer 21

Depends partly on the semaphorin - neurolipin signalling

Question 22

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.

Question 23

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

Question 24

How can you examine the role of NFPC and TAF1 in axonogenesis if negative constructs results in inhibited axonogenesis?

Answer 24

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

Question 25

What is the appropriate control this experiment - looking at the pathways of axons which have negative constructs that affect their development

Answer 25

Axons electroporated with an myc vector or a vector containing GFP only.

Question 26

What is the effect of replacing NFPC and TAF1 with NF∆E-myc or TAF1∆N-myc?

Answer 26

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

Question 27

What is another method that was used to knock out NFPC and TAF1?

Answer 27

We used antisense morpholino oligonucleotides (morpholinos) as a further approach to knock down NFPC and TAF1 function

Question 28

How would you evaluate efficient knockdown by the morpholinos?

Answer 28

Western blot analysis

Question 29

What is the purpose of a western blot?

Answer 29

Detect specific proteins in a sample of tissue homogenate or extract.

Question 30

What are the stages of a western blot?

Answer 30

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.

Question 31

What are anti-sense morpholino oligonucleotides?

Answer 31

Morpholino oligos are advanced tools for blocking sites on RNA to obstruct cellular processes.

A Morpholino oligo specifically binds to its selected target site to block access of cell components to that target site. This property can be exploited to block translation, block splicing, block miRNAs or their targets, and block ribozyme activity.

Question 32

After electroporation of the fluorescently labeled morpholinos into the retina, how were the RGC axons visualised?

Answer 32

Labelling with the lipophilic tracer Dil at stage 40

Question 33

What was used as a control for NFPC-MO- or TAF1-MO?

Answer 33

Axons from retinas loaded with control morpholino

Question 34

What was the activity of Con-Mo?

Answer 34

Showed a robust normal trajectory to the tectum

Question 35

What was the activity of NFPC-MO- or TAF1-MO axons?

Answer 35

Often short and many appeared to stall or strayed anteriorly, particularly in the MOT segment around the caudal turn

Question 36

The experiment uses two techniques to cause loss of function: dominant negative and morpholino knockdown. However there was a discrepancy in the percentage of brains that had axons entering the tectum when using these two techniques. How does the author account for this discrepancy?

Answer 36

Flourescent morpholino signal was too small to visualise. To combat this they scored all the Dil labelled axons.

RGC axons were visualised by anterograde labelling with the lipophilic tracer Dil.

The issue with this as that not all of these axons are morpholino loaded

The myc tag used in the dominant negative technique allowed identification and exclusive scoring of dominant negative–expressing axons.

Question 37

After disruption of the NFPC and TAF1 signalling where do most of the axon defects occur?

Answer 37

Around the MOT

Question 38

What was the activity of the NF∆E:eGFP-expressing axons?

Answer 38

Advanced through the optic tract at a significantly slower average rate than GAP-GFP control axons

AND

30% of NF∆E:eGFP axons (7 of 23 axons) stalled at the caudal turn in the MOT, which was not observed in control samples

Thus, axons expressing dominant negative NFPC grew at normal speeds through the VOT but slowed or stalled at the caudal turn, supporting the idea that NFPC function is particularly important in this mid-to-dorsal segment of the optic tract.

Question 39

How did the authors study the importance of NFPC cell-cell recognition?

Answer 39

They designed a blocking domain consisting of the NFPC ectodomain, this was fused to an Fc fragment (antibody constant domain).

This NFPC:Fc fragment was introduced into the intact optic pathway before the entry of retinal axons using an exposed brain preparation

Question 40

What is the FC region?

Answer 40

Fragment crystallizable region

is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system.

Question 41

How were the axons visualised in the cell-cell recognition experiment?

Answer 41

retinal axons were then antero- gradely labeled with DiI at stage 40

Question 42

What are the useful properties of Dil?

(pronounced dye aye)

Answer 42

Diffuse laterally to stain the entire cell

Fluoresce weakly in water but highly fluorescent and quite photostable when incorporated into membranes

Possess very bright signals with high extinction coefficients

Are well retained in cell membranes

Demonstrate very little transfer to other cells

Question 43

What was the effect of NFPF:Fc treated treated brains?

Answer 43

Exhibited pathfinding defects in the mid-to-dorsal optic tract.

The defects included stalling at the caudal turn as well as is-projection phenotypes such as misrouting anteriorly into the telencephalon bypassing the caudal turn and failing to reach the tectum

Only 65.9% of NFPC:Fc brains had axons that entered the tectum, in comparison to 100% of brains incubated with Con-Fc

Question 44

Experiments to control for specificity showed that the treatments did not disrupt the expression pattern of the known key optic tract guidance cues Slit1b and Sema3A

What is the significance of this?

Answer 44

Slit 1, Slit 2 and Semaphorin 3A demarcate the rostral and dorsal boundaries and prevent axons from entering the telencephalon. So the results seen could be explained by a change in function of these molecules, therefore by checking that there is no disruption in the expression of Slit1b and sema3A you are making sure that the results are purely from the NFPC:Fc protein.

Question 45

‘but the phenotypes seen with NFPC:Fc above do not distinguish whether the pathfinding defects are due to axon- axon or axon-substrate interactions’

Answer 45

This is a limitation of the NFPC:Fc experiment

Question 46

How did the authors test whether NFPC function is required in the neuroepithelial cells of the optic pathway substrate?

Answer 46

we disrupted NFPC function exclusively in the optic tract neuroepithelium by targeted delivery of NFPC and TAF1 morpholinos (NFPC- MO or TAF1-MO, respectively) into the cells of the presumptive optic pathway (diencephalon) at stage 29/30 by electroporation.

At stage 40, we labeled untransfected retinal axons with DiI to visualize their trajectories through the electroporated substrate. Morpholino-loaded areas of optic tract neuroepithelium were identified by green fluores- cent signal.

Question 47

What was the effect of loading NFPC and TAF1 morpholinos into the presumptive optic pathway (diencephalon) by electroporation?

(This experiment was to find out if errors in pathfinding by NFPC were due to axon-axon or axon-substrate interactions, so this test was to remove functioning NFPC in the neuroepithelium substrate)

Answer 47

Electroporation of Con-MO into the optic pathway sub- strate did not affect the pathfinding of the retinal axons to the tectum (Fig. 5b). In contrast, NFPC- and TAF1-depleted substrate pathways produced axon pathfinding errors similar to those that occurred with knockdown exclusively in retinal axons (Fig. 5c,d). Most of the retinal axons in Con-MO brains grew normally through morpholino-loaded areas, whereas a large fraction of NFPC-MO or TAF1-MO brains had retinal axons that failed to grow through morpholino-loaded regions of the optic pathway (Fig. 5e and Supplementary Table 1). Similarly, the proportion of brains in which retinal axons misprojected out of the optic tract or bypassed the caudal turn altogether (data not shown) was considerably higher in NFPC-MO (78.4%; P < 0.001, Fisher’s exact test) and TAF1-MO (80.8%; P < 0.001, Fisher’s exact test) than in Con-MO brains (4.5%). Finally, the proportion of brains having retinal axons that stalled at the caudal turn was significantly higher in NFPC-MO and TAF1-MO brains than in Con-MO (Fig. 5e). Together with the NFPC-Fc blocking-peptide results above, this sug- gests that NFPC-mediated interactions between retinal axons and their neuroepithelial substrate are crucial for pathfinding in the mid- to dorsal optic tract.

Question 48

What are dominant negative genes?

Answer 48

They produce an altered gene product that acts antagonistically to the wild-type allele

Question 49

In NF∆E-myc, what does the myc stand for?

Answer 49

Myc is a family of regulator genes and proto-oncogenes that code for transcription factors

Question 50

Where is sema3a mRNA expressed strongly?

Answer 50

in a domain adjacent and rostral to the MOT at the caudal turn

Question 51

How did the authors deplete endogenous sema3a?

Answer 51

Blastomere injection and targeted forebrain electroporation of sema 3A morpholinos

Question 52

What was the result of Blastomere injection and targeted forebrain electroporation of sema 3A morpholinos?

Answer 52

ectopic projections of retinal axons

Question 53

What might explain the relatively low penetrance (only 30% of Sema3A morphants showed at least one axon navigating away) ?

Answer 53

The relatively low penetrance may reflect a combination of mosaic MO expression, incomplete Sema3A knockdown and cue redundancy

Question 54

How did the authors perturb the sema3A signalling in vivo?

Answer 54

They used a function blocking anti-body against neurolipin - 1

(alternative strategy)

Question 55

What is the significance of the fact that Sema3A and NP-1 depletion result in similar stalling and misprojection phenotypes in the optic tract.

Answer 55

Suggests that these molecules may work together to help guide retinal axons through the caudal turn into the dorsal optic tract

Question 56

What is the response of growth cones to sema3A application?

Answer 56

Appreciable growth cone collapse within 5 min, increasing to a maximal response of 60% at 10 min followed by substantial recovery by 60 min (data not shown). Notably, we found a rapid and marked increase in growth cone NFPC immunofluorescence after Sema3A exposure as compared to that in control growth cones exposed to vehicle alone (Fig. 6a,b and Supplementary Fig. 5). This cue-induced NFPC response was specific to Sema3A, as it did not occur with Slit2 or lysophosphatidic acid (LPA) (Fig. 6c,d,f), two other cues that also cause retinal growth cone collapse within 10 min.

After semaphorin application, NFPC was observed in the filopodia. However this peripheral staining is rarely seen in unstimulated growth cones.

By 60 min after Sema3A exposure, when most growth cones had recovered from collapse, NFPC immunoreactivity had dropped and moved back into the central domain.

Question 57

‘This cue-induced NFPC response (increase in NFPC) was specific to Sema3A, as it did not occur with Slit2 or lysophosphatidic acid (LPA) (Fig. 6c,d,f), two other cues that also cause retinal growth cone collapse within 10 min’

What is the significance of this?

Answer 57

This shows that the Sema3A-induced NFPC increase was cue-specific rather than a general feature of reti- nal growth cone collapse mechanisms.

The increase in NFPC was a trait specifically of semaphorin as opposed to generic growth cone collapse

Question 58

How did sema3a affect TAF1?

Answer 58

Resulted in a similar increase and distribution

Question 59

How does NP-1 change with sema3a application?

Answer 59

Notably, as seen previously, NP-1 showed the opposite change, a rapid downregulation followed by recovery, suggesting that NFPC is not acting in a receptor complex with NP-1 for Sema3A.

Question 60

How can we inhibit sema3A growth cone repulsive responses?

Answer 60

Sema3A-induced growth cone repulsive responses can be blocked by inhibitors of protein synthesis and that Sema3A elicits rapid protein synthesis

Question 61

Quantitative immuno- fluorescence analysis showed that the rapid increase in growth cone NFPC was completely blocked by the translation inhibitors anisomy- cin (Fig. 7b) or cycloheximide (Supplementary Fig. 5n), but not by the transcription inhibitor 􏰁-amanitin.

Account for this

Answer 61

Transcription inhibitors block the formation of mRNA from DNA. However there may already be free mRNA in the cell and therefore translation and protein expression may still occur in these cells.

? perhaps

Question 62

What is the purpose of the kaede protein?

Answer 62

Allows visualisation of newly synthesised green proteins within minutes

Question 63

Sema3A and NFPC both act to promote axon progression past the caudal turn, but how can we determine if the sema3a induced translation of NFPC is region specific?

Answer 63

we expressed Kaede-NFPC-3’UTR in navigating retinal axons and monitored Kaede-green signal after photoconversion with time-lapse microscopy at the VOT or MOT—that is, either before or at the caudal turn

Question 64

The authors detected no increase in the native Kaede-green signal in growth cones in the VOT (0 of 7 growth cones; Fig. 8a,f and Supplementary Video 3); however, there was a significant increase in the native Kaede-green signal in growth cones in the MOT (5 of 10 growth cones; Fig. 8b,f and Supplementary Video 4), indicating de novo synthesis of the reporter.

How can we tell if this increase in NFPC is as a result of Sema3A?

Answer 64

To test whether this region-specific increase in NFPC translation reporter activity is regulated by Sema3A, we used the NP-1 function-blocking antibody in vivo to perturb the interaction of Sema3A with its receptor. When we applied the antibody in vivo, we observed significantly less Kaede-green signal recovery after photoconversion in growth cones in the MOT than when we applied a control antibody

Question 65

What are the roles of protocadherins?

Answer 65

Formation of axon projections through the regulation of outgrowth, dendritic arborization, spine number26, synaptogenesis and neuronal survival

And in this study - axon steering

Dendritic arborization, also known as dendritic branching, is a multi-step biological process by which neurons form new dendritic trees and branches to create new synapses

Question 66

How does NP-1 and classical CAM L1 affect the function of Sema3A?

Answer 66

ligand binding induces direct associations between NP-1 and the classical cell adhesion molecule (CAM) L1, altering the Sema3A receptor complex itself and thereby modifying subsequent Sema3A-elicited axon behaviors

NP-1 ligand binding with L1 results in changes in the Sema 3A receptor complex. Sema3a causes the endocytosis of the NP-1-L1 complexes leading to decreased axon-axon fasiculation which is critical for formation of the anterior commisure.

The anterior commissure (also known as the precommissure) is a white matter tract (a bundle of axons) connecting the two temporal lobes of the cerebral hemispheres across the midline, and placed in front of the columns of the fornix.

Question 67

Why is it unlikely that the sema3a receptor participates in NFPC function?

Answer 67

Sema3A stimulates a loss of NP-1 simultaneously with the NFPC increase

Question 68

What is the relationship that typically exists between the strength of adhesion and the speed of axon growth?

Answer 68

Typically considered an inverse relationship (when strength of adhesion increases the speed of axon growth decreases)

The finding that NFPC depletion caused stalling in the MOT and slowed growth toward the tectum in the dorsal optic tract may therefore seem puzzling.

However, although highly adhesive substrates do impede axon growth in vitro, the highest growth rates are observed at intermediate levels of adhesivity

Question 69

The discussion says that NFPC axon-substrate interactions influence the formation of the optic tract - how can this be inferred?

Answer 69

We found that retinal axons strayed from their normal trajectories when NFPC was depleted in the substrate alone before axon entry

Question 70

Do protocadherins always work homophilically?

Answer 70

No, certain activity-dependent protocadherins also work heterophilically with classical cadherins, as has been demonstrated with arcadlin26 and OL-protocadherin41

Question 71

In the NFPC-poor VOT, where growth cones advance rapidly, the translation reporter activity was low. When growth cones reached the NFPC-rich MOT, where they slow and turn, NFPC translation reporter activity markedly increased.

translation reporter activity is referring to the kaede protein that is attached to the 3’ UTR region of Xenopus NFPC

Question 72

What other transmembrane cue receptor molecules are upregulated at specific locations as a result of local translation?

Answer 72

EphA2 and Robo3

Question 73

What is cofilin?

Answer 73

An actin binding protein, which disassembles actin filaments

Question 74

Give examples of specific guidance cues that can trigger the translation of specific mRNA subsets

Answer 74

For example, the translation of 􏰃-actin (actb) mRNA in the growth cone is required for turning toward the attractant netrin-1 (refs. 14,17), and repulsive Slit2 stimulation induces the synthesis of the actin depolymerizing protein cofilin5,15. Similarly, NFPC mRNA is locally translated in response to Sema3A but not to Slit2.

Question 75

Why did The NP-1–blocking antibody diminish but did not totally abolish the translation of Kaede- NFPC-3􏰂UTR in vivo.

Sema3a said to stimulate the translation of NFPC, NP-1 is the cofactor necessary for the function of sema3a

Answer 75

This may be because the anti-NP-1 is not capable of fully inhibiting the Sema3A in this assay or because there may be other cues at the caudal turn that could also activate NFPC translation

Question 76

What other mRNA of proocadherins are present in growth cones?

Answer 76

mRNAs encoding several different protocadherins (for example, pro- tocadherins alpha11, beta, gamma3, 9, 14) are also found in retinal growth cones, raising the possibility that their local synthesis may modulate other specific aspects of axon pathfinding and synapse formation

Question 77

What happens to TAF1 mRNA when there is semaphorin3a?

Answer 77

coordinated translation-dependent upregulation of TAF1

This is in keeping with TAF-1’s key role as a key effector of NFPC function

TAF1, first described as a transcriptional regu- lator45, may act in the RGC nucleus before retinal axon initiation12. However, as translation but not transcription is required for Sema3A chemotropic responses13, TAF1 is likely to act nontranscriptionally at the growth cone in regulating NFPC adhesive signaling.

So increase in translation of TAF-1 and corresponding non-transcriptional regulation of NFPC adhesive signalling

Question 78

Where are classical axon repellants semaphorin and slit found?

Answer 78

Sema3A and Slit, classical axon repellents, are expressed in adjacent, nonoverlapping domains anterior to the developing optic tract near the caudal turn

Question 79

Why is it thought that semaphorin and slit ‘give a push from behind’?

Answer 79

If these molecules acted solely as classical repellents, their depletion would be predicted to cause axons to stray into their normal domains of expression. Although some axons do exhibit these types of errors in Sema3A- and Slit1-depleted brains, many axons stall unexpectedly at the caudal turn3. This stalling pheno- type suggests that these molecules not only act as repellents but also give a “push from behind” to promote growth

Question 80

Main finding

Answer 80

Our results suggest that Sema3A, by upregulating axonal NFPC through local translation, helps promote axon growth in the NFPC-rich mid- to dorsal segment of the optic tract and thereby facilitates accurate navigation along the final segment of the route to the tectum. We suggest that the upregulation of specific adhesion molecules in growth cones in response to local diffusible cues could be used as a general mechanism to couple pathway experience to appropriate interactions for the journey ahead.