Complete samenvatting van de tutor lessen in course 10, studie jaar 3. De samenvatting is per week geschreven en is dus gebaseerd op de stof die behandeld is per les. De samenvatting is volledig en bevat alle stof die men dient de leren voor het C. elegans gedeelte in de thematoets. In deze samenva...
Tutor assignment 1
The assignments, which you need to execute during your research for Professor
Woollard are composed of theoretical and practical parts and will help you to become
an expert in using C. elegans to study the molecular principles of cancer development.
Tutor Task 1: In the tutor class 1, discuss the different model organisms that are currently
used for studying cell divisions. Perform a literature study of the model organisms and their
advantages and disadvantages in researching (a)symmetric cell divisions. In the following
tutor class, present your findings in a mind map for the different model systems.
1. How do I culture C. elegans in order to perform reliable experiments?
Different strains of C.elegans are used (AW673 & JR667), culture for 2 or 3 days, check the
worms and transfer to a new plate to maintain the stock and keep them alive. Gravid worms
can be used for bleaching to synchronize the worms (get L4’s, for further experiments).
NGM+OP50 is used for feeding, the OP50 E.coli has a resistance marker for streptomycin.
Based on the resistance marker bacteria are selected that can grow on the NGM. OP50 is
made weak it cannot produce any uracil. Make it reliable by using the same conditions
(changing the parameters can have unexpected effects on the worms, try to use different
batches of worms to exclude any inter assay variations).
2. What is the best way to maintain the worms?
Put the worms on NGM plate with OP50 to feed from, incubate for 3 days. When transferring
worms transfer 3 (L4) worms to a new plate. Temperature for incubation should be between
15 °C and 25 °C.
3. Can I identify the different stages of a worm?
The L4 worm is easy for everyone to identify. The L2 and L3 are harder to observe, but not
necessary to identify.
Tutor Task 2: Study the article of Dr Samantha Hughes and Professor Woollard (Hughes et
al. (2013) Biology Open 2(7):718). Make sure that you understand the critical experiments
and the main conclusions of the research findings in this paper as this paper forms the
theoretical basis of the C. elegans research. Based on this paper, you should answer the
questions below. Discuss your answers in tutor class 2 and generate a model answer sheet,
which you need in preparation for the theme test. The consultation with the expert will
consider the paper in more detail which will be helpful to complete your answers to these
questions below.
1. What is the key characteristic of a stem cell?
Stem cells can divide asymmetrically. They have the ability to self-renew, and thereby remain
a stem cell, or produce daughter cells that can differentiate. Multiple cell types can be
derived from single self-renewing stem cells, and because of that they have the potential to
generate all tissue types. So, capable to produce one daughter that adopts a differentiated
fate and another that remains a stem cell and proceeds to proliferate further. Cell
differentiation is the process whereby a little specialized cell turns into a more specialized
cell. Proliferation is cell division (cell proliferation = letterlijk vermeerdering van cellen).
2. What are C. elegans seam cells?
A seam cell is a hypodermal blast cell that produces lateral cuticular ridges (alae) during the
L1 larval and adult stages. Seam cells divide during the larval stages to produce more seam
cells, hyp7 cells — the main hypodermal (skin) cell for the body — and neurons (in a subset
of seam cell lineages). Seam cells are arranged as longitudinal rows of 16 cells on the left
and right sides of the body. Seam cells divide in the stem-like mode throughout larval
development and divide asymmetrically, giving rise to an anterior daughter that fuses with
the hypodermis and a posterior daughter that proliferates further (they are not normal stem
cells, just stem cell like because they have a terminal differentiation). So, neuroectodermal
seam cell; asymmetric divisions in order to both self-renew and differentiate into epidermal
cells, neurons, glia and ray precursor cells of the male tail.
, 3. How can you visualise seam cells in C. elegans?
The seam cell can be visualized by attaching a reporter gene to the nuclei. An example of a
reporter gene is GFP (green fluorescent protein). These nuclei then will glow green under the
fluorescence microscope. In the article they used GFP constructs under control of a seam
specific promotor (bro-1CNE).
4. There are two possible cell types resulting from the division of a mother seam cell. What
are they? Think about the V1-4 lineage in particular.
The asymmetric division results in; a posterior daughter which retains the ability to divide
further; an anterior daughter which differentiates, most commonly contributing to epidermal
tissue by fusing with the hyp7 syncytium. V1-V6 are anterior daughter cells.
5. Draw a diagram to show the general pattern of symmetric and asymmetric seam cell
divisions in C. elegans.
6. Rnt-1 is the single C. elegans Runx homolog. Why is it important in (a)symmetric
divisions?
The Runx transcription factor rnt-1 and its DNA binding partner bro-1 (a homologue of the
Runx binding factor CBFb) are crucial to regulate the balance between seam cell proliferation
and differentiation. rnt-1 is expressed in the proliferated daughter cell.
7. What was the purpose of the genome-wide RNAi screen?
To identify new regulators of seam cell divisions, this plays a role in balancing the
proliferation and differentiation (if the polarity is lost in the cell division it can cause a
cancerous phenotype).
8. Two genes were identified from the screen. What were they and what is the effect of their
removal on the worm?
Found ceh-20 and unc-62 (encoding TF: Pbx and Meis), they Regulate the proliferation of
seam cell. Knockdown caused severe hyperplasia (seam cell number is doubled) of seam
cells resulting from symmetrisation of divisions that are normally asymmetrically. So, CEH-
20/Pbx and UNC-62/Meis were identified. Both are TALE-class transcription factors. Animals
lacking these genes show seam cell hyperplasia restricted to the anterior of the worm.
9. What alleles of unc-62 and ceh-20 were shown to have the most significant effect on seam
cell number?
CEH-20: Mu290 increased seam cell number AND Ay9 hyperplasia. Unc-62: ku234 (mutant
allele): stronger phenotype AND e644: no effect on seam cells. They have a significant
effect on mainly the anterior side of C.elegans seam cell number.
The ku234 allele results in a point mutation in the start codon of the 1b transcript. This has
been suggested to shift the start codon downstream, likely resulting in a truncated protein
lacking the important Pbx interacting HM domain. So, in unc-62 the start codon is normally
ATG. In the ku234 allele a point mutation occurred in ATG (= methionine). If Taq polymerase
comes along it keeps going on until it reaches the first methionine. So this leads to an
alternative start, it is not a frameshift mutation! Because of the alternative start, it isn’t known
where it will start. It shifts to somewhere downstream and it isn’t sure if a functional protein is
made. Alternative splicing occurs (figure). Unc-62 has eight exons, two of them are
alternative (exon 1 and exon 7).It could be that exon 1a is made and that 1b is skipped. It
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