XB-IMG-138199
Xenbase Image ID: 138199
|
Fig. 6. Disruption of RA signaling relocates
TRPP2 away from the plasma membrane. (A) RA
disruption in lateral mesoderm does not affect pkd2
expression. Isolated LMZ explants were taken from
embryos previously injected with GFP mRNA
(control) or with a mix of GFP and Cyp26 mRNA,
and cultured until siblings reached the early neurula
stage (NF stage 14) for RT-QPCR analysis.
Mean6s.e.m. results from three independent
experiments are shown. Although disruption of RA
signaling causes a strong inhibition of pax8 and
lhx1 expression, pkd2 expression is not
significantly affected. *P,0.05; **P,0.01 (paired
Student’s t-test) (B) Schematic representation of
TIRF microscopy. TIRF microscopy exclusively
images signals arising close to the cell membrane.
TIRF works by directing excitation light through a
glass substrate towards an aqueous specimen at
an angle to obtain total internal reflection due to the
refractive index decrease at the glass–water
interface. In these conditions, an evanescent wave
is created in the liquid with the same wavelength as
the incident light. This evanescent wave decreases
exponentially with distance. The wave is able to
excite fluorophores only near the interface. It
provides an ‘optical sectioning’ effect similar to, but
even narrower, than that achieved by a confocal
microscope (Axelrod, 2008). (C) Representative
views of the imaging field for a hTRPP2–GFP
mRNA-injected explant (left) and for an explant coinjected
with hTRPP2–GFP mRNA and Cyp26
mRNA (right). (D) Histogram (mean6s.e.m.)
displaying the calculated area of GFP spots per
field of view (50650 mm) in control kidney fields
(hTRPP2–GFP) and in kidney fields
overexpressing the RA-catabolizing enzyme
Cyp26. 350 and 700 pg of Cyp26 mRNA was
injected. Disruption of RA signaling induces the
relocation of TRPP2 proteins away from the plasma
membrane. This effect is dose-dependent. Image published in: Futel M et al. (2015) Copyright © 2015. Image reproduced with permission of the Publisher, The Company of Biologists Ltd. Larger Image Printer Friendly View |