First published online March 7, 2005
doi: 10.1242/10.1242/dev.01726
Development 132, 1737-1748 (2005)
Published by The Company of Biologists 2005
Dorsoventral patterning of the Xenopus eye: a collaboration of Retinoid, Hedgehog and FGF receptor signaling
Giuseppe Lupo1,*,
Ying Liu2,*,
,
Rong Qiu2,
Roshantha A. S. Chandraratna3,
Giuseppina Barsacchi4,5,
Rong-Qiao He2 and
William A. Harris1,
1 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2
3DY, UK
2 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, PR China
3 Retinoid Research, Departments of Chemistry and Biology, Allergan, Irvine, CA
92623, USA
4 Dipartimento di Fisiologia e Biochimica, Universita' degli Studi di Pisa, Via
Carducci 13, 56010 Ghezzano (Pisa), Italy
5 AMBISEN Center, High Technology Center for the Study of Environmental Damage
to the Endocrine and Nervous System, Universita' degli Studi di Pisa, Pisa,
Italy
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Fig. 1. Effects of Hh signaling overexpression on eye DV polarity. (A,B) The red or
light-blue ß-gal staining identifies the injected side. The broken yellow
circles highlight the eye region. (A) Unilateral injection of low doses of
bhh mRNA (1 pg) upregulates Vax2 in the DR and reduces
ET in stage 33 Xenopus embryos. Pax2, Vax1b and
Raldh3 are partially upregulated, but not in the DR, and the
Pax6 domain is slightly reduced in these embryos. On the uninjected
side, Vax2 is normally expressed in the OS and the VR; ET is
expressed in the DR; Pax2, Vax1b and Raldh3 are expressed in
the OS region and Pax6 is expressed in the retina region. (B) High
doses of bhh mRNA (250 pg) upregulate Vax2, Pax2, Vax1b and
Raldh3 throughout the eye, and repress ET and Pax6
expression. (C) Schematic representation of the results shown in A and B. Low
Hh levels partially expand the OS and ventralize in part the DR. Purple
indicates the overlap of ventral and dorsal character in the most dorsal
retina. High Hh levels transform the entire retina into OS.
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Fig. 2. Effects of RA signaling overexpression on eye DV polarity. (A) Low RA doses
(0.1 µM) expand Pax2, Vax1b and Raldh3 expression
domains, but do not significantly affect Vax2- and
ET-positive regions in stage 33 Xenopus embryos.
Pax2-hybridized embryos are also shown in frontal view (right
column). (B) High RA doses (10 µM) upregulate Vax2 in the DR and
repress Vent2, Pax2, Vax1b and Raldh3, but do not change
Pax6 expression. (C) Schematic representation of the results shown in
A and B. Low RA levels enlarge the OS. High RA levels ventralize the retina
and repress OS formation.
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Fig. 3. Effects of FGFR signaling overexpression on eye DV polarity. (A) Unilateral
injection of 2 pg iFGFR1 mRNA, followed by induction with AP20187 at
stage 12.5/13, partially expands the expression domains of Vax2, Pax2,
Vax1b and Raldh3 in the ventral eye, and weakly represses
ET and Pax6 in stage 33 Xenopus embryos. (B)
Injection of 4 pg iFGFR1 mRNA upregulates Vax2, Pax2, Vax1b
and Raldh3 in the dorsal eye and strongly repress ET and
Pax6. (C) Schematic representation of the results shown in A and B.
Increasing FGFR levels progressively expands the OS at the expense of the
retina.
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Fig. 4. Expression pattern of Raldh3 during Xenopus development.
(A) Raldh3 expression as detected by whole-mount in situ
hybridization of whole embryos (stage 10.25-31) or dissected neural retinas,
without lens and pigmented epithelium (stage 59). cf, choroid fissure. Scale
bar: 350 µm. (B) Raldh3 expression in transverse sections of stage
33 embryos after whole-mount in situ hybridization. From left to right,
sections show expression at progressively posterior levels. op, olfactory
placode; os, optic stalk. (C) Comparison of Pax2, Raldh3 and
Vax2 expression in the optic cup in transversal sections of stage 33
embryos after whole-mount in situ hybridization. (D) Double in situ
hybridizations of Rx1 with Raldh2, Shh or FGF8 on
mid- to late neurula embryos shown from anterior view. The inset shows a
double in situ hybridization of Rx1 with FGF8 on an early
neurula embryo.
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Fig. 5. Raldh3 overexpression in the presence of ATR reproduces the effects of RA
treatments on eye DV polarity. (A) Embryos were bilaterally injected with a
total of 1 ng Raldh3 mRNA and treated with 0.5 µM ATR from stage
12.5/13, followed by molecular marker analysis at stage 33. The combination of
Raldh3 and ATR expands Pax2 expression, while ATR alone has only a
weak effect. A comparable expansion of Pax2 is obtained by a dose of
2 µM ATR in the absence of exogenous Raldh3. (B) The combination of 4 ng
Raldh3 mRNA and 2.5 µM ATR can upregulate Vax2 in the DR,
while ATR alone has only a weak effect. Strong Vax2 upregulation is
also caused by a dose of 10 µM ATR in the absence of exogenous Raldh3, as
shown in both lateral (upper row) and anterior view (lower row).
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Fig. 9. RA, Hh and FGFR signaling pathways can crossregulate each other at the
transcriptional level. (A) High dose RA treatments (10 µM) strongly repress
Shh expression in the anterior midline (indicated by the yellow
bracket) both at neurula and stage 33. The same RA doses cause fusion of the
two stripes of FGF8 expression in the ANR and the anteroventral
ectoderm in neurula stage embryos, while at stage 33 FGF8 expression
is strongly downregulated in the whole head region. Low dose RA treatments
(0.1 µM) have weaker effects on both Shh and FGF8. (B)
Unilateral injection of 500 pg bhh mRNA laterally expands
FGF8 expression in the anterior neural ridge but does not affect
Raldh2 expression at neurula stages. The inset shows FGF8
expression in the ANR of an uninjected embryo at the same stage. The same dose
of bhh upregulates both FGF8 and Raldh2 in the eye
region at stage 33. (C) Unilateral injection of 4 pg iFGFR1 mRNA,
followed by induction with AP20187 at stage 12.5/13, expands Shh
expression in the prospective hypothalamic region at neurula, and activates
Shh in the eye regions at stage 33. At doses of 4 and 2 pg,
iFGFR-1 mRNA has no significant effect on Raldh2 expression
at neurula stages and stage 33, respectively.
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Fig. 6. RA, Hh and FGFR signaling pathways can partially ventralize the eye field
at neurula stages. (A) In embryos treated with 10 µM RA from stage 12.5/13
(right column), the lateral region of Vax2 domain (indicated by the
yellow brackets) is expanded at late neurula stages with respect to control
embryos, while Vent2 domain is reduced. (B) Mid-late neurula embryos
unilaterally injected either with 500 pg bhh mRNA or with 2.5 pg
iFGFR1 mRNA, followed by induction with AP20187 from stage 12.5/13.
Compared with the uninjected side, Vax2 expression is expanded, while
ET is repressed. The broken yellow lines indicate the embryo
midline.
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Fig. 8. FGFR and Hh signaling can collaborate in eye ventralization. (A) Embryos
were unilaterally co-injected with 1 pg bhh mRNA and 0.5 pg
iFGFR1 mRNA, and induced with AP20187 from stage 12.5/13, followed by
molecular marker analysis at stage 33. Pax2 is mainly activated
within the ventral half of the eye (class I eyes) by bhh alone, while
in the presence of iFGFR1 and bhh Pax2 expression extends
into the dorsal eye (class II and class III eyes). Co-injection of
bhh and iFGFR1 mRNAs also slightly increases the frequency
of eyes with ubiquitous Vax2 expression and more strongly represses
Tbx5 when compared with bhh alone. Under these conditions,
iFGFR1 mRNA alone has no obvious effect on Pax2, Vax2 and
Tbx5 expression. (B) Quantification of the effects of bhh
and iFGFR1 on Pax2, Vax2 and Tbx5 expression.
Between 24 and 30 embryos were analyzed for each sample in these experiments.
(C) Schematic representation of the results shown in A. In class II eyes, the
expanded OS does not reach the most dorsal retina, which can acquire in part
ventral identity. In class III eyes, the retina is mostly or completely
transformed into OS.
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Fig. 7. RA and Hh signaling can collaborate in eye ventralization. (A) Embryos were
unilaterally injected with 1 pg bhh mRNA and treated with 2 µM ATR
from stage 12.5/13. Pax2 is strongly upregulated in the eye by the
combination of bhh and ATR, but weakly by bhh or ATR alone
in stage 33 Xenopus embryos. Vax2 expression can be
activated in the DR by these doses of bhh in the absence of ATR.
ET is reduced by bhh or ATR alone, but is strongly repressed
by the combination of bhh and ATR. (B) Embryos were unilaterally
injected with 0.2 pg bhh mRNA and treated with 5 µM ATR from stage
12.5/13. Vax2 is upregulated in the DR by the combination of
bhh and ATR, but not by bhh or ATR alone. These doses of
bhh and ATR weakly activate Pax2 expression. ET
expression in the DR is partially reduced by bhh or ATR alone, but is
strongly repressed by the combination of bhh and ATR. The yellow
arrow indicates ET expression domain in the DR. (C) Quantification of
the effects of bhh and ATR on Pax2, Vax2 and ET
expression. Between 21 and 25 embryos were analyzed for each sample in these
experiments. (D) Schematic representation of the results shown in A and B. The
combination of 1 pg bhh mRNA with 2-2.5 µM ATR strongly expands
the OS. The combination of 0.2 pg bhh with 5 µM ATR ventralizes
the DR.
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Fig. 10. Loss-of-function effects of RA, Hh and FGFR signaling pathways on eye DV
polarity. Embryos were treated from stage 10.5 with 10 µM AGN194310 (AGN),
100 µM cyclopamine (CPM) and 25 µM SU5402 (SU) in different
combinations, and analyzed for molecular marker expression at stage 30/31. (A)
Effects of the single inhibition of any of the RA, Hh and FGFR signaling
pathways, when compared with mock-treated embryos. (B) Effects of double and
triple inhibition of RA, Hh and FGFR pathways. (C) Schematic representation of
the results shown in A and B. Strong eye dorsalization is caused by triple
inhibition of RA, Hh and FGFR signaling, while double inhibitions produce
weaker effects.
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Fig. 11. A proposed model for signaling events controlling DV patterning of the eye.
Interaction of high levels of Hh and FGFR with low levels of RA signaling
repress Pax6 and induce the expression of Vax1, Pax2 and
Vax2, leading to OS specification. High levels of RA in collaboration
with low levels of Hh and FGFR signaling repress ET and induce the
expression of Vax2 in the absence of Vax1 and Pax2,
but in the presence of Pax6, thus specifying VR identity. BMP
signaling inhibits Vax2 and induces ET expression, causing
DR specification. In addition, high levels of RA repress the expression of Hh
and FGFR signals, while Hh and FGFR signaling crossactivate each other at the
transcriptional level.
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© The Company of Biologists Ltd 2005
