The
advantages of C. elegans as a research tool are well established,
being a multicellular eukaryotic organism that is simple enough to be
studied in great detail. From a practical perspective it is small enough
to be compatible with microbeam irradiation and a wide variety of
mutants and transgenics are readily available, as is a large community
of C. elegans researchers. C. elegans is particularly well
adapted to microbeam irradiation due to its thin body, allowing particle
penetration to targets throughout the entire animal with minimal beam
scattering. The abundance of genetically modified worms with fluorescent
markers provides an easy way to target individual organs and quantify
the required endpoint. Currently we can irradiate either chemically
anesthetized C. elegans worms, in our standard microbeam dish, or
mechanically constrained worms in a microfluidic channel.
The first RARAF microbeam irradiation studies with
C. elegans were recently published. This
study used C. elegans strain SJ4005 hsp-4::gfp(zcls4)V, which has
a GFP reporter for the hsp-4 heat-shock gene. Heat shock proteins (HSPs)
are a ubiquitous family of gene products present in cells under
unstressed conditions that are expressed in much higher concentration in
the presence of stress. HSP responses can be induced by diverse
stressing agents including heat, UV irradiation, γ-rays irradiation and
chemicals.
In this study, young adult C. elegans
hermaphrodites were used. Before exposure, worms were individually
imaged using an epifluorescent microscope. For
microbeam irradiations, worms were anesthetized and placed in a
customized microbeam dish with a micro cover-slip for individual
exposure. Animals were irradiated at the tail, in the center of the GFP
expression region, with 0, 25, 50, and 75 3-MeV protons (LET 12.5 keV/µm,
max. penetration 140 µm). The
microbeam diameter was 1
µm. (This is indicated by a red dot on the figure below). Control worms
were mock-irradiated, by targeting the microbeam just outside the worm
(~ 200 μm) and using the same set-up time for anesthetic exposure and
concentrations. After exposure, the micro cover-slips were removed using
a micro aspirating pump, and worms were washed with buffer and
re-cultured in standard agar/covered Petri dishes. Based on well
established protein kinetic studies and our preliminary expression time
studies, we selected a twenty four hour time point for GFP expression
evaluation.

Microbeam exposed worms showed a different GFP stress
response in terms of intensity and localization, compared with the
control group. Increase in expression was noted only after delivering 50
and 75 protons. Mock irradiated worms did not exhibit any GFP stress
response after 24 hours (Fig. a). No apparent stress response was
detected when 25 protons were delivered (Fig. b). However, when the
worms were exposed to 50 or 75 protons, a strong stress response in the
posterior intestine was observed, between the spermatheca and the
microbeam targeted area (Fig. c-d). A one micrometer diameter proton
beam was able to induce tail region in situ GFP over-expression
as well as distal stress response as far as >150 µm away from the
irradiated spot. No stress response was seen in other regions of the
body of exposed worms, and only basal levels of GFP expression were
detected at the pharynx and spermathecae regions.
Sugimoto
previously used wild type and a mutant strain C. elegans as an
in vivo model for microbeam irradiation. However, in those
experiments C. elegans were irradiated with a collimated ~ 20-50
µm diameter microbeam of 220 MeV carbon particles (LET of 120 keV/µm).
Worms were exposed to 1500 carbon ions delivered to an extended worm
body area. While these irradiation techniques are useful to study organ
or region exposures, they do not allow the exposure of a restricted
number of cells or small tissue sections in a living organism. Our
technique is capable of delivering, with high accuracy, small numbers of
particles at the sub-cellular and cellular level. Many phenomena under
study using microbeams, such as the bystander phenomenon, must involve
cell-to-cell communication, extracellular environment, and functional
integrity. The capability to target individual tissue components is
quite compelling for such studies.
We show that low doses of protons delivered at the
tail region, where different organs and cell populations are located,
are capable of inducing both local and distal GFP over-expression. This
response is remarkable since the microbeam used is estimated to traverse
a series of spatially co-localized cell structures. How this spatially
restricted stressor is capable of inducing a distal stress response is
not understood. However, it is worth noting that among the organs
potentially irradiated are the intestine and the stomato-intestinal
muscle. Cell-cell communications as well as tissue damage signaling are
potential mechanisms involved in these responses.
Belyakov et al suggested
that autocrine/paracrine mechanisms or juxtacrine signaling are
potential bystander pathways to explain long range bystander phenomena
in complex tissues. Future studies will be focused on the identification
of cell targets and signal mechanisms using different biological
endpoints.
Use of this targeted irradiation method provides a
new tool to investigate complex long-range biological responses such as
the bystander effect in living organisms. Future developments include a
microfluidics-based worm clamp for microbeam irradiation without
anesthesia as well as microbeam modulation techniques to adjust the
position of the Bragg peak to different locations within the target
organism.
More detail can be found
in A. Bertucci, R.D. Pocock, G. Randers-Pehrson, and D.J. Brenner.
Microbeam irradiation of the C. elegans nematode. J. Radiat.
Res. (Tokyo) 50 Suppl A: A49-54 (2009).
The
medaka (Oryzias latipes) is a small egg-laying freshwater fish,
which is increasingly being used as model system for developmental
genetics and evolutionary biology. The medaka embryo is particularly
well suited for microbeam studies, due to its small size and
optical clarity for imaging internal tissues and organs. The medaka
genome has recently been sequenced, and many transgenics and mutants are
now available. We have focused on the medaka rather than zebrafish,
because it is somewhat more hardy, but essentially all the irradiation
protocols are directly applicable for zebrafish.
We have incorporated an online
system for Medaka embryo orientation into the microbeam end station.
This allows targeting organs, by using their position related to
prominent physiological features (e.g. the eyes), which are easily
visible using our microbeam imaging system.