THESIS
2012
xxi, 146 p. : ill. (some col.) ; 30 cm
Abstract
Using the bioluminescent Ca
2+ reporter, f-aequorin, in conjunction with
luminescence microscopy, I have identified both short-range fast (i.e., travelling at ~49
μm/sec over a distance of ~80 μm) and long-range slow (i.e., travelling at ~4 μm/sec over a
distance of ~235 μm) propagating Ca
2+ waves that are generated in the external yolk
syncytial layer (E-YSL) of developing zebrafish embryos. Embryos were microinjected with
f-aequorin into the top of the yolk cell at the 128-cell stage in order to preferentially load the
Ca
2+ reporter into the E-YSL as it formed and developed. These two classes of Ca
2+ waves
begin during the late blastula period, between the dome stage and 30% epiboly, and
continue for ~90 minutes (i.e., until the germ ring/shield stage). The majority of both cla...[
Read more ]
Using the bioluminescent Ca
2+ reporter, f-aequorin, in conjunction with
luminescence microscopy, I have identified both short-range fast (i.e., travelling at ~49
μm/sec over a distance of ~80 μm) and long-range slow (i.e., travelling at ~4 μm/sec over a
distance of ~235 μm) propagating Ca
2+ waves that are generated in the external yolk
syncytial layer (E-YSL) of developing zebrafish embryos. Embryos were microinjected with
f-aequorin into the top of the yolk cell at the 128-cell stage in order to preferentially load the
Ca
2+ reporter into the E-YSL as it formed and developed. These two classes of Ca
2+ waves
begin during the late blastula period, between the dome stage and 30% epiboly, and
continue for ~90 minutes (i.e., until the germ ring/shield stage). The majority of both classes
of waves are generated in the dorsal quadrant of the embryo and move in both directions
around the E-YSL. The E-YSL was identified as the site of Ca
2+ wave generation by: (i)
combined luminescence/fluorescence imaging of embryos injected with f-aequorin and
SYTOX Green, the latter being used to specifically label the yolk syncytial nuclei (YSN);
and (ii) multi-photon imaging of embryos loaded with the fluorescent Ca
2+ reporter, Calcium
Green-1 dextran. I also investigated the localisation of the YSN and endoplasmic reticulum
(ER) in the E-YSL during the early stages of epiboly when the E-YSL Ca
2+ waves are
known to be actively propagating, and subsequently proposed a hypothesis that involves
YSN clustering, and their associated peri-nuclear ER, as well as a requirement for a short
inter-nuclear distance for effective E-YSL Ca
2+ wave generation and propagation. In
addition, treating embryos with a Ca
2+ chelator (5,5’-dibromo BAPTA), or an IP
3R
antagonist (2-APB) resulted in the inhibition of the E-YSL Ca
2+ signals and a subsequent
delay in late epiboly. On the other hand, treatment with antagonists of RyRs (ryanodine and
dantrolene) had no significant effect on the Ca
2+ signals or on development. Morpholino
knockdown of regulator of G-protein signalling 3 (rgs3) reproducibly and reliably
suppressed the E-YSL Ca
2+ waves and led to a delay in late epiboly, as well as
dorsalisation of embryos. The possible roles of Rgs3 in the regulation of Ca
2+ signalling in
the E-YSL, and in zebrafish development are discussed.
Post a Comment