Kubke Lab:/Notebook/Cranial nerve development/2015/01/06
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Today I had another long think about the direction in which I was headed and I clarified a lot in my head. I realised that I need to take advantage of the things I already know I can do well rather than try and master new skills within the next 5 weeks, not only for the sake of time but also for the sake of my own enjoyment. That basically put the idea of trying to dissect stage 14 embryos out of the picture and in doing so, my initial hypothesis can't really properly be answered in the way that I thought it might be to begin with. That is probably one of the biggest lessons I've learnt so far - that things change as you learn more and try more and I need to embrace that and realise it's part of the scientific method.
What I have decided to focus on is the point at which efferents turn to exit the alar plate to the nerve. It occurs in r3 and r5 very obviously and at certain ages is a very obvious and sharp almost 90 degree turn. I had initially thought that the afferents might be touching them and making contact with them to help them to know when to turn, but the literature suggests that at least for the 7th nerve that is not the case as the efferents appear to exit and hence turn before afferents have even entered.
I thought at first that I could just image the turn at different ages and see if I noticed any patterns or consistencies in where and when they were turning. I thought that there were a couple of different hypotheses for things that may be stimulating the efferents to turn.
1. that it was encouraged by afferents,
2. that it was at the alar/basal plate boundary and that there was an intrinsic repulsive factor at that point that caused the turn
3. that there was a chemotactic factor produced by either the rhombomere in which the exit point is contained or by the exit point itself
4. that there was something about the r3 and r5 rhombomeres that signalled to the nerves that they needed to turn up
5. that the boundary at which they turned (the lateral motor boundary) had a repulsive factor (not that it was the alar/basal plate boundary because I don't know that yet)
I talked to Fabiana around lunch and she suggested that I increase the 'resolution' of my question by actually dying different peripheral nerves of the same bigger nerve (eg V2 and V3 with DIO vs V1 with DiI of the trigeminal nerve) and then look at the arrangement of the cell bodies within the hindbrain that correlate to each peripheral nerve. Her feeling was that although the cell bodies were highly organised into their discrete bundles in the adult, they were not like this in the embryo and were infact all mixed up. I could then analyse the trajectory of the turn that the different axons make and see if there is any reason to believe that neurons that contributed to different peripheral nerves, moved in a different way to other peripheral nerves.
In this way, I am still going to be answering my question of what could be a mechanism by which Branchiomotorneurons (BMN) leave the hindbrain but through a more specific hypothesis.
This afternoon I dissected a stage 26 embryo and dyed the 2 different branches of the trigeminal nerve different colours. That will be exciting to see tomorrow if I can get two different coloured axons within the same rhombomere in the hindbrain! Hope it works out
Also, serendipitously, Martin Wild, a colleague of Fabiana's (and mine I guess) alerted her to an article about the segregation of the trigeminal nerve components in the hindbrain in a stage 33 chicken embryo, showing that the cell bodies were organised and clustered together with the neurons that would end up in the same peripheral nerve as them. So it will be interesting to see if I can observe the process (by doing different stages and reconstructing a time series) of the cells going from less arranged to moving into discrete bundles that correspond to their future peripheral nerve.