How do neural stem cells wake up? An investigation of

potentially involved molecular signals

A summer research project carried out by James Butler in Dr Claudia Barros’ laboratory in June 2015

1. Aims:

1. Validate the expression of potential novel players in neural stem cell (NSC) mitotic reactivation.

2. Contribute to experiments testing the function of a particular transcript found upregulated in

active versus dormant NSCs and currently under investigation in the laboratory.

2. Expression analysis; methods and results: 2.1 Primer design

I designed primers for three gene candidates arising from the

microarray screen: Mts, GCKIII and Rheb.

Primers designed mainly on the computer using websites such as

Primer3 and Flybase.

Parameters such as primer size, primer Tm and GC% must be kept

within strict limits.

2.2 Polymerase chain reaction (PCR)

Used to test the primers I designed.

Involves mixing complementary DNA with nucleoside triphosphates,

H2O, a pH buffer, and DNA polymerase.

PCR machine then cycles the samples through temperature changes

to stimulate artificial DNA replication.

The primers I designed failed to show significant bands, however the

control pairs did work.

2.3 Gel electrophoresis

2% agarose gel used to test the primers designed previously, following PCR

Primers I designed failed to show significant bands

The control pair and the ladder were successful and did show, suggesting that the problem was with the designed primers.

Had this stage been successful, the primers would have been used in quantitative real-time PCR reactions.

3. Contribution to functional analysis of Dmob4; methods and results

3.1 Fly husbandry

Drosophila melanogaster NSCs are used because of their 1. proven

genetic and morphological similarities with human NSCs, and 2. their

well-defined NSC lineages that can be followed and genetically

manipulated in vivo with single-cell resolution.

I learned the general lab maintenance tasks involved with using

Drosophila, such as making fly food and washing bottles and vials.

I also learned how to distinguish the sex of the flies and check

whether females are virgins visually.

I collected female virgins of genotype UAS-dicer2; Repo-GAL4

Planned a cross aimed at knocking down dmob4 using a UAS-dmob4-

RNAi male line.

The Repo-GAL4 line drives expression in glia cells.

3.2 Staging Drosophila larvae for dissection at precise ages

The virgin females and males of the genotypes mentioned above were

crossed.

Crossed parental strains were then added to chambers and allowed to

lay for one hour.

Eggs would then have been left to develop until they reached 18 hours

post hatching.

Number of eggs laid was insufficient and so this project had to be

completed later by Ellie Gonzaga.

3.3 Larval brain dissection

Larval brains are dissected in a rubber-bottomed dish using

microdissection forceps and fine scissors.

After an initial incision 1/3 from the head tip, the tissues surrounding

the brain are stripped away to expose the ventral nerve cord and

pair of optical lobes. The brains can then be stained.

4. Conclusion

Even though I was not able to see my project through to the end, I

gained lots of experience in the laboratory environment.

I learned and practiced core skills and techniques and was able to reach

a level of competence that meant I was able to work alone.

In future I will allow myself more time to carry out projects in order to

ensure I always have time to complete it and minimise the impact of

problems along the way.

GAL4/UAS system for targeted gene

expression

GAL4 used to stimulate transcription under

Upstream Activating Sequence (UAS)

control.

RNAi is used to inhibit gene expression.

The GAL4 and UAS elements are in separate

parental lines, so phenotypic effects occur

only in the progeny.

Figure 1: Phelps & Brand, 1998, “Ectopic Gene Expression in Drosophila using GAL4 system”.

Figure 2: Rossi & Gonzalez, 2015

Figure 1: Homem &

Knoblich, 2012. Drosophila

neurogenesis occurs in two

waves, with a quiescent

‘G0’ stage in between. Dr

Barros performed a single-

cell microarray screen

comparing transcriptomes

of dormant to mitotically-

active NSCs to reveal

potential novel players in

the reactivation process.