First published online July 21, 2003
doi: 10.1242/10.1242/dev.00626
Targeted expression of the dominant-negative FGFR4a in the eye using Xrx1A regulatory sequences interferes with normal retinal development
Li Zhang1,
,
Heithem M. El-Hodiri1,,*,
Hai-Fei Ma3,
,
Xue Zhang4,
Marc Servetnick5,
Theodore G. Wensel4 and
Milan Jamrich1,2,3,
1 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
2 Departments of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
3 Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
4 Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
5 Department of Biology, Ithaca College, Ithaca, NY 14850, USA
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Fig. 3. Schematic diagram of the constructs used to make transgenic
Xenopus tadpoles for the characterization of Xrx1A
regulatory sequences. Transgene construct 1 was made by cloning the
Xrx1A regulatory sequences (SstI-PstI fragment) in
front of GFP in pBS-GFP. The full-length promoter construct
1 was digested with NotI and SalI, AvaI,
BamHI, BglII, and BanI, respectively, to release
the transgene constructs 2, 3, 4, 5 and 6. Transgene construct 9 was obtained
by inserting a heat shock protein promoter (hsp) into the SmaI site
of pBS-GFP. The BglII-BanI fragment from the
Xrx1A promoter was subcloned into the EcoRV site of
construct 9 and pBS-GFP to generate transgene constructs 7 and 8,
respectively. To make transgene constructs 12 and 13, the
SstI-BamHI fragment from the Xrx1A promoter was
subcloned into pBS-GFP first, then the hsp promoter (blunt-ended
HindIII-NcoI fragment of phs3LSN) or the
BanI-PstI fragment from the Xrx1A promoter were
inserted into the EcoRV site between the Xrx1A early
enhancer and GFP. Construct 10, containing nucleotides (nt) -857 to 0
of the Xrx1A regulatory sequence, was prepared by PCR of construct 1,
with a GFP-specific primer (see below) and the Xrx1A promoter
specific primer: 5'-GATCGGATCCCTTCCAGCAATCATATCCTA-3' (-857 to
-838). The resulting product was digested with PstI (3'-end of
the Xrx1A regulatory region) and BamHI (included in the
Xrx1A-specific primer) and subcloned into pBS-GFP. Construct
11, including nt -986 to -838 of the Xrx1A regulatory region was
prepared by PCR of construct 1 using the following primers:
5'-GATCAGATCTTAGGATATGATTGCTGGAAG-3' (the complement of the
previous primer encompassing nt -857 to -838, but with a BglII site
at the end); and 5'-GATCGGATCCGATCTGTTATCTGGAAAACCCC-3' (nt -986
to-965 of the Xrx1A regulatory sequence and a BamHI site).
The PCR product was digested with BamHI and BglII and
subcloned into the BamHI site of construct 9.
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Fig. 1. Transgenic Xenopus laevis embryos at different stages carrying
Xrx1A-GFP construct 1 (A,D,G,J; see
Fig. 3), displaying GFP
fluorescence (B,E,H,K). (C,F,I,L) In situ hybridization of Xrx1A
probe to non-transgenic embryos of the same developmental stage to demonstrate
the normal expression pattern of the Xrx1A gene. A-C, stage 15; D-F,
stage 21 (frontal view); G-I, stage 21 (side view); and J-L, stage 28.
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Fig. 2. (A) Section of a stage 40 transgenic Xenopus laevis embryo
carrying Xrx1A-GFP construct 1 (see
Fig. 3) and displaying GFP
fluorescence in photoreceptor cells. (B) High magnification of a section
through an eye of a stage 40 transgenic tadpole shows fluorescence in the
photoreceptor layer. (C) The same section stained using antibodies against
rhodopsin. (D) Overlap of B and C, demonstrating that the rhodopsin-positive
rods (yellow cells) express GFP. However, some rhodopsin-negative cells also
express GFP (arrowhead). (E) Additional staining with Topro-3 visualizes other
retinal cells. (F) High magnification of a section through an eye of stage 40
transgenic tadpole displays fluorescence in the photoreceptor layer. (G)
Staining of the same section with antibodies against calbindin, a marker of
cone cells. (H) Overlap of F and G, demonstrating expression of GFP in cone
cells (yellow cells). (I) Additional staining of the same section with Topro-3
visualizing other retinal cells.
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Fig. 5. (A-C,N-R) Immunostaining of sections of tadpole eyes with antibodies
against rhodopsin and calbindin. (D-M) Whole-mount staining of tadpoles with
antibodies against rhodopsin. (A) Section of a stage 39 tadpole that does not
carry the Xrx1A- FGFR4a construct stained with
antibodies against rhodopsin, demonstrating the presence of photoreceptor
cells. (B) Section of a stage 39 tadpole that carries the
Xrx1A-FGFR4a construct stained with antibodies
against rhodopsin. Note the lack of photoreceptor cells. (C) Section of a
tadpole carrying the Xrx1A-FGFR4a construct stained
with rhodopsin antibodies that shows some photoreceptor cells in ectopic
position. (D-G) Whole-mount staining of Xenopus tadpoles that do not
carry the Xrx1A-FGFR4a construct with rhodopsin
antibodies at different stages, demonstrating the normal accumulation of
photoreceptor cells during development. (H-M) Whole-mount staining of
transgenic Xenopus tadpoles expressing the
Xrx1A-FGFR4a construct with rhodopsin antibodies at
different stages, demonstrating lower numbers of photoreceptor cells in these
embryos at all stages. D,H, stage 33; E,K, stage 35; F,L, stage 36; G,M, stage
38. (N) Staining of a section from a stage 46 embryo that does not carry the
Xrx1A-FGFR4a construct with antibodies against
rhodopsin. (O) Staining of a section of stage 45 embryo that does not carry
the Xrx1A-FGFR4a construct with antibodies against
cone-specific calbindin. (P) An eye section from a stage 45 tadpole expressing
the Xrx1A-FGFR4a construct stained with rhodopsin
antibodies. Note the lack of rhodopsin-positive rods. (R) An eye section from
a stage 45 tadpole expressing the Xrx1A-FGFR4a
construct stained with calbindin antibodies. Only few cones are present
(arrowheads), some of them in ectopic locations.
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Fig. 7. Comparison of retinal cell distribution in tadpoles carrying and lacking
the Xrx1A-FGFR4a construct. (A) Immunostaining of an
eye section from a stage 45 non-transgenic tadpole with antibodies against
Islet1, which recognizes the ganglion and amacrine cells. (B) Immunostaining
of an eye section from a stage 45 tadpole that carries the
Xrx1A-FGFR-4a construct with antibodies against
Islet1, demonstrating disturbed layering of retinal cells. (C) Hoechst
staining of the section from B. (D) Immunostaining of an eye section from a
stage 45 tadpole that does not carry the Xrx1A-FGFR4a
construct with antibodies against glutamine synthetase, which recognizes
Müller cells. (E) Immunostaining of an eye section from a stage 45
tadpole that carries the Xrx1A-FGFR4a construct with
antibodies against glutamine synthetase demonstrates irregular distribution of
Müller cells in the retina of these tadpoles. (F) Hoechst staining of the
section from E. (G) Histogram showing the percentage of Müller glial
cells and retinal ganglion cells/amacrine cells in the retina of transgenic
tadpoles. Müller glial cells and retinal ganglion cells/amacrine cells
are identified by immunostaining with antibodies against glutamine synthetase
and Islet1, respectively. MGC, Müller glial cells; RGC, retinal ganglion
cells; AC, amacrine cells; Single Tsg, car-GFP transgenic (MGC,
n=8 retinas; RGC/AC, n=6 retinas); Double Tsg,
car-GFP/Xrx1A-FGFR4a transgenic (MGC, n=10
retinas; RGC/AC, n=11 retinas).
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© The Company of Biologists Ltd 2003
