Direct visualization of organ morphogenesis and
function using the zebrafish embryo
Nathan D. Lawson, Ph.D.
Program in Gene Function and Expression
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Tuesday, June 19, 2012
Using the zebrafish as a model system
Tuesday, June 19, 2012
Tuesday, June 19, 2012
Rapid early development:
The first 18 hours in 30 seconds
Rolf Karlstrom, UMass Amherst
Tuesday, June 19, 2012
Rapid early development:
Organogenesis in a day (or two)
beginning at 16-20hpf:
nervous system
neurons, glia, neural crest, etc. .
digestive system
intestine, liver, etc.
endocrine system
pancreas, pituitary, thyroid. .
cardiovascular system
blood vessels, heart, blood
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Tuesday, June 19, 2012
Transparent embryos and external development
Tuesday, June 19, 2012
Visualizing blood vessels in zebrafish embryos
endothelial
cells
lumen
angiography
Tg(fli1:egfp)y1
fli1:egfp
Tuesday, June 19, 2012
Transparency and transgenics = detailed
analysis of vascular anatomy in a live embryo
Tuesday, June 19, 2012
Imaging modalities applied to zebrafish in the Lawson Lab
Transmitted brightfield
Leica MZ6 and MZ12
dissection microscopes
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Tuesday, June 19, 2012
Imaging modalities applied to zebrafish in the Lawson Lab
Epifluorescence
Leica MZFLIII and M165
fluorescence microscopes
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Tuesday, June 19, 2012
Imaging modalities applied to zebrafish in the Lawson Lab
differential interference contrast
microscopy (DIC/Nomarski)
Zeiss Axioskop2 /
water immersion lenses
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Tuesday, June 19, 2012
Imaging modalities applied to zebrafish in the Lawson Lab
Confocal microscopy
- Leica SP2
- 5 laser lines: 405, 458, 488, 561, 633
- 3 detectors
double fluorescent in
situ hybridization using
antisense riboprobes
Tuesday, June 19, 2012
live double transgenic
Notch indicator line /
all endothelial cell
live transgenic subjected to
angiography
Notch indicator line /
quantum dots
Imaging modalities applied to zebrafish in the Lawson Lab
2-photon microscopy
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Tuesday, June 19, 2012
What is 2-photon excitation?
- excitation using higher wavelength photons
(than single photon)
- longer wavelength with femtosecond interval
pulses to achieve necessary activation energy to
stimulate emission
Tuesday, June 19, 2012
Why use 2-photon excitation?
- longer wavelength light is less damaging to
biological specimens
- longer wavelength penetrates deeper into
tissue than confocal (up to 1 mm)
Tuesday, June 19, 2012
Why use 2-photon excitation?
2-photon versus confocal microscopy
- excitation only at focal plane
- all emitted photons can be
captured = NO pinhole
Tuesday, June 19, 2012
Zeiss 7MP
Microscope
• Zeiss Axio Examiner
• equipped with upright motorized
X-, Y-, Z-stage
• 20x and 40x IR water-immersion
lenses (NA=1.0)
• equipped with temperature
controlled, light-tight chamber
equipped for whole embryo imaging
Computer/software
• 1 TB storage
• standard off-the-shelf Zeiss software (ZEN)
• multi-time time lapse plug-in to allow for
parallel multisample image acquisition
Post-processing
Imaris
ImageJ
Tuesday, June 19, 2012
Zeiss 7MP
Laser source
Chameleon Ultra II (Coherent, Inc.)
• tunable between 690 to 1080 nm
• power @ 920 nm >1.6W, @ 1080 >200 mW
• tuning speed: >40 nm/s
capable of:
1. exciting blue, green, red fluorescent
proteins
2. rapid wavelength switching
Detectors
non-descanned (i.e. no pinhole)
- high sensitivity
capable of simultaneous detection of 3
separate emission spectra using fixed
filter cubes
Tuesday, June 19, 2012
- live zebrafish embryos mounted in low-melt agarose
- w/ “tricaine” anesthetic and PTU (prevents pigmentation)
- time-lapse at 28°C, ambient air environment
- longer term (>12 hours) requires recirculating system
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Tuesday, June 19, 2012
Published applications of 2-photon imaging
in collaboration with Clive Standley and Kevin Fogarty:
Siekmann et al. (2009) Chemokine signaling guides regional patterning of
the first embryonic artery. Genes and Development, 23:2272-7.
- in vivo time lapse of deep tissue vessels
Nicoli et al. (2010) microRNA-mediated integration of haemodynamics
and Vegf signaling during angiogenesis. Nature, 464:1196-200.
- in vivo time lapse of deep tissue vessels
using Lawson Lab 7MP:
Zhu et al. (2011) Evaluation and application of modularly assembled zinc
finger nucleases in zebrafish. Development, 138:4555-4564.
- analysis of vascular morphology in live wild type and knockout
transgenic embryos
Nicoli et al. (2012) miR-221 is required for endothelial tip cell behaviors
during vascular development. Developmental Cell, 22:418-429.
- in vivo time lapse imaging of angiogenesis in double transgenic embryos
Tuesday, June 19, 2012
Aortic endothelial cells have distinct origins and
morphogenetic behaviors
anterior lateral
dorsal aorta
dorsal aorta
posterior lateral
dorsal aorta
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Siekmann et al. (2009) Genes and Development, 23:2272-7.
Tuesday, June 19, 2012
cxcr4aum20 mutant embryos fail to form lateral dorsal aortae
wild type
cxcr4a mutant
Siekmann et al. (2009) Genes and Development, 23:2272-7.
Tuesday, June 19, 2012
mir-221 is required for endothelial tip cell proliferation and
migration during angiogenesis
Tg(fli1a:negfp)y7;(fli1ep:dsredex)um13
~75 micron deep stack (3 micron sections; 512x512)
acquired every 15 minutes for ~12 hours
Nicoli et al. (2012) Developmental Cell, 22:418-429.
Tuesday, June 19, 2012
miR-221 modulates proliferation and PI3K output to drive tip
cell behavior in the Flt4 signaling pathway
kdrl / flt4 vegfc
cdkn1b
miR-221 Dll4 pik3r1
vegfa
Notch
miR-221
dorsal aorta
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cardinal vein
Gerhardt et al. (2003) J. Cell Biol. 161, 1163–1177
Siekmann & Lawson (2007) Nature 445, 722–726
Hellstrom et al. (2007) Nature 445, 776–780
Nicoli et al. (2012) Developmental Cell, 22:418-429.
Tuesday, June 19, 2012
flt4
cdkn1b
pik3r1
Multispectral imaging
- application of “Brainbow” fluorescent proteins for
long term lineage tracing
Tg(kdrl:cre);(ubi:Brainbow1.1)
Aurelie Quillien, unpublished
Tuesday, June 19, 2012
Multispectral imaging
- application of photoconvertible proteins to assess temporal
control of signaling events during embryonic development
photoconversion
@ 36 hpf
image
@ 60 hpf
Tg(tp1:kaede)
Kaede - photoconverted at early developmental stage
Kaede - not converted, at stage of image acquisition
angiography with fluorescent microspheres
Aurelie Quillien, unpublished
Tuesday, June 19, 2012
Application of 2-photon to in vivo imaging
Benefits
• lower phototoxicity
• better optical sectioning
• increased sensitivity
• enables long term in vivo imaging
Drawbacks
• expensive
• still potential toxicity due to heat
• low resolution (both time and space)
- space options: STED
- time options: SPIM (sheet illumination)
• limited user group
• still challenging to image some RFPs
Tuesday, June 19, 2012
Acknowledgements
UMass Medical School, Worcester
Program in Gene Function and Expression (PGFE)
Lawson Lab
Ann Grosse
Fatma Kok
George Kourkoulis
Ira Male
John Moore
Chih-Wen Ni
Aurelie Quillien
Samir Sissaoui
Sarah Sheppard
Masahiro Shin
Tom Smith
Jacques Villefranc
ubi:brainbow
Albert Pan
Alex Schier
Tg(kdrl:cre)
Neil Chi
Tuesday, June 19, 2012
Bioinformatics
Abirami Lakshmanan (Lawson Lab)
Julie Zhu (PGFE)
Former:
Stefania Nicoli
Yale CVRC
Arndt Siekmann
MPI Münster
Biomedical Imaging
Group
Clive Standley
Kevin Fogarty