First published online 26 January 2005
doi: 10.1242/dev.01673
Development 132, 913-923 (2005)
Published by The Company of Biologists 2005
Distinct developmental programs require different levels of Bmp signaling during mouse retinal development
Deepa Murali1,2,*,
Shunichi Yoshikawa1,*,
Rebecca R. Corrigan1,2,
Daniel J. Plas3,
Michael C. Crair3,
Guillermo Oliver4,
Karen M. Lyons5,
Yuji Mishina6 and
Yasuhide Furuta1,2,
1 Department of Biochemistry and Molecular Biology, University of Texas, M.D.
Anderson Cancer Center, Houston, TX 77030, USA
2 Program in Genes and Development, Graduate School of Biomedical Sciences
(GSBS), University of Texas-Houston, Health Sciences Center and M.D. Anderson
Cancer Center, Houston, TX 77030, USA
3 Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030,
USA
4 Department of Genetics, St Jude Children's Research Hospital, Memphis, TN
38105, USA
5 Departments of Molecular, Cell and Developmental Biology, Orthopedic Surgery,
and Biological Chemistry, University of California, Los Angeles, CA 90095,
USA
6 Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health and Safety/NIH, Research Triangle Park, NC 27709,
USA
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Fig. 1. Bmpr1a function is not required for embryonic retinal development.
Control samples are from Bmpr1a+/fx;Cre mice and
mutants are from Bmpr1a-/fx;Cre mice. (A)
Hematoxylin and Eosin stained sections of the adult retina from 3-month-old
mice. Retinal lamination pattern and cell types are unaltered in the
Bmpr1a-/fx;Cre mutants (right), compared with
control littermates. (B) Anterograde tracing of retinal ganglion cell axons
into the superior colliculus of the midbrain of control (left) and mutant
(right). Fluorescent images of the left half of the midbrain of 9-day-old pups
that have received a focal injection of DiI into the right dorsal retina,
arrow indicates termination zone of dorsal axons. The axes in the left
superior colliculus are indicated; a, anterior; l, lateral; m, medial; p,
posterior. (C) Schematic representation of the Bmpr1a genomic locus
and various mutant alleles. (D) Genomic PCR to detect Bmpr1a alleles
using primers indicated in C. The genotypes of the sample DNAs are indicated
at the top. In the retina of adult mice carrying the Cre transgene, the
amplicon for the conditional allele (fx) is undetectable (arrowhead),
whereas in the tail the PCR product corresponding to the unrecombined
conditional allele is readily detected. Consistent with recombination of the
floxed allele occurring only in the retina, the dE2 amplicon, which
represents the recombined allele with exon 2 removed, is present only in the
retina of mice carrying the Cre transgene. gcl, ganglion cell layer; inl,
inner nuclear layer; onl, outer nuclear layer; rpe, retinal pigment
epithelium.
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Fig. 3. High levels of Bmp signaling activity, principally mediated by Bmp4,
tightly regulate Tbx5 expression in the dorsal retina. (A,B)
Whole-mount in situ hybridization for Tbx5. Lateral views of E9.0-9.5
embryos (A) and E10.5 embryos (B) probed for Tbx5 transcripts. (A)
Dorsal specific Tbx5 expression is lost in the optic vesicle of an
advanced Bmp4-/- mutant embryo (arrowhead). (B)
Tbx5 transcripts in the dorsal retina (arrowheads) are detected at
comparable levels in both control (top) and
Bmpr1a-/fx;Bmpr1b+/-;Cre
mutant (bottom) samples at E10.5. Tbx5 transcripts are also strongly
expressed in the forelimb buds (fl) in both control and mutant. (C) In situ
hybridization on coronal sections of E11.5 embryos with dorsal towards the
top. Tbx5 expression is lost by E11.5 in the mutant retina (bottom,
arrowhead). (D) Immunohistochemistry for P-Smad1/5/8 detects high levels of
P-Smad protein in the dorsal retina of control embryos, defining regions
receiving the highest Bmp signal (top). This pattern is lost in mutants
(bottom). so, somites; fl, forelimb; le, lens; nr neuroretina; rpe, retinal
pigment epithelium. Scale bar: 500 µm in A; 1 mm in B; 100 µm in
C,D.
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Fig. 2. Retinal dorsoventral patterning defects in
Bmpr1a-/fx;Bmpr1b+/-;Cre
mutant mice. Control littermates shown are representatives of the
Bmpr1a+/fx;Bmpr1b+/+;Cre or
Bmpr1a+/fx;Bmpr1b+/-;Cre
genotypes. (A) The Bmpr1a-/fx;
Bmpr1b+/-;Cre mutants show no overt morphological
defects in the eye. (B-E) Hematoxylin and Eosin-stained sections (B),
ß-TubulinIII antibody staining for retinal ganglion cells (C),
anti-PKC for bipolar cells (D) and anti-Syntaxin for amacrine cells (E)
reveal that the retinal lamination pattern and cell types are unaltered in the
Bmpr1a-/fx;Bmpr1b+/-;Cre
mutants (bottom) compared with control littermates (top). (F) Analyses of
retinotectal axon projection in the superior colliculus of the midbrain in
postnatal day 7 (P7) animals. Focal injection of DiI into the dorsal retina of
the left eye reveals a single termination zone in a lateral-posterior region
of the contralateral (right) superior colliculus in normal animals (top,
arrowhead). By contrast, in the mutants, several ectopic termination zones are
seen (bottom, arrows), in addition to a normal lateral-posterior spot (bottom,
arrowhead). The axes in the right superior colliculus are indicated: A,
anterior; L, lateral; M, medial; P, posterior. (G-J) Coronal sections of
embryonic eyes with dorsal towards top. In the mutants, the expression of
dorsal markers Efnb2 (G) and Tbx5 (I) is lost, while
transcripts of Ephb2 (H) and Vax2 (J), which are normally
ventrally enriched, are now expanded throughout the developing retina (H,J).
gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; nr, neuroretina;
prl, photo receptor layer. Scale bar: 500 µm in A,G,H; 100 µm in
I,J.
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Fig. 4. Asymmetric expression of Raldh genes in the retina is maintained in
Bmpr1a-/fx; Bmpr1b+/-;Cre
mutants. In situ hybridization on coronal sections of E12.5 embryos with
dorsal towards the top. Arrows indicate the domains of expression within the
retina. (A,C) The transcripts for genes encoding retinoic acid synthesizing
enzymes Raldh1 (Rldh1a1) (A) and Raldh3 (Rldh1a3) (C) are
asymmetrically expressed in the retina with high levels dorsally and
ventrally, respectively. In addition, they are also expressed at low levels in
the surface ectoderm surrounding the eye region (A,C), the lens (A) and the
dorsal RPE (C). (B,D) The expression pattern is not significantly altered in
the Bmpr1a-/fx;Bmpr1b+/-;Cre
mutant embryos. le, lens; nr, neuroretina; rpe, retinal pigmented epithelium;
se, surface ectoderm. Scale bar: 100 µm.
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Fig. 5. Defects in retinal growth in
Bmpr1a-/fx;Bmpr1b-/-;Cre
double mutant mice. (A) Side views of neonates. The double null mutant has an
anophthalmic phenotype (bottom). (B) Side views of the eyes of E12.5 embryos.
Reduced eye size and incomplete closure of the retinal pigmented epithelium
ventrally are seen in the double mutant (bottom). (C) Coronal sections of
embryos shown in B. (D) Cell-death analyses at E11.5 indicate a significant
increase in TUNEL-positive cells (green) in the mutant retina (counterstained
with propidium iodide). (E-G) Although there is no significant change in the
percentage of BrdU-positive cells up to E11.5 (green/yellow cells) (E,G),
there is a rapid reduction of cell proliferation by E12.5 (F,G; broken line in
F outlines the degenerating mutant retina; a relatively large error bar in the
E11.5-12.5 mutant group reflects a spectrum of phenotypic severity during
these stages). le, lens; nr, neuroretina. Scale bar: 500 µm in B; 200 µm
in C; 100 µm in D-F.
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Fig. 6. Bmpr1a-/fx;Bmpr1b-/-;Cre
double null mutants display a range of defects in the expression of retinal
marker genes. Analyses of gene and protein expression in coronal sections of
embryonic retina at the indicated stages by radioactive in situ hybridization
(A,C,D), DIG in situ hybridization (E,F,G) or immunostaining (B,H). (A) At
E11.5, expression of Chx10 is attenuated in the mutant retina (right)
compared with control. (B) CyclinD1 is specifically absent in the mutant
retina (right), whereas protein expression in the adjacent lens tissue is
maintained. (C,D) Lhx2 and Rx, two early pan-retinal
markers, are expressed in the mutant retina at comparable levels with the
control, despite the apparent degenerative phenotypes at this stage. (E) The
mutant retina fails to initiate retinal neurogenesis at E11.5 as indicated by
the loss of Math5 expression. Arrows indicate comparable
Math5 expression both in the diencephalons of both control and
mutant. This is not simply a developmental delay, as Math5 is absent
even at E12.5 (not shown). (F) Brn3b, a downstream target of Math5
expressed in the newly born ganglion cells in the central retina, fails to be
induced in the mutants. (G) Expression of Pax6 is maintained in the
mutant. (H) Neuronal tubulin expression is detected in the mutant retina. le,
lens; nr, neuroretina; rpe, retinal pigment epithelium. Scale bar: 100
µm.
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Fig. 7. Fgf15 is a potential downstream target of Bmp signaling in the
retina. (A) Fgf15 is strongly expressed in the distal optic vesicle
at E9.0-9.5 in a wild-type embryo. (B) The expression of Fgf15 is
absent in the eye (arrowhead) of an advanced Bmp4-/-
mutant embryo with equivalent number of somites to the wild-type embryo shown
in A. (C,D) Application of BMP4-soaked beads (asterisks) restores the
expression of Fgf15 in the distal optic vesicle of
Bmp4-/- by 18 hours in culture (C, arrowhead). Under the
same condition, BSA fails to restore the expression of Fgf15 (D,
arrowhead). Arrows indicate the future retinal pigmented epithelium slightly
displaced anteriorly during explant culture. (E) Fgf15 expression
persists through later stages as shown for an E11.5 normal retina
(Bmpr1a+/fx;Bmpr1b+/-;Cre
control). (F) In
Bmpr1a-/fx;Bmpr1b-/-;Cre
double mutant, the expression of Fgf15 is absent (arrows). le, lens;
ov, optic vesicle. Scale bar: 50 µm in A-D; 100 µm in E,F.
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Fig. 8. A model for the function of Bmp signaling through Bmpr1a/Bmpr1b
receptors in the retina. (A) Schematic representation of an early embryonic
eye, indicating the sources of Bmp ligands. Black dots represent dorsally
localized Bmp4 transcripts
(Furuta and Hogan, 1998 ),
arrows indicate Bmp7 from the lens ectoderm
(Wawersik et al., 1999) and
arrowheads indicate Bmp2 signaling from the retinal pigmented epithelium
(Dudley and Robertson, 1997).
(B) Expression of the corresponding Bmp receptors in the eye. Bmpr1a
is ubiquitously expressed (hatching), whereas Bmpr1b shows a graded
expression within the retina with highest levels ventrally (dots). (C) A
relative distribution Bmp signaling activity along the retinal dorsoventral
(DV) axis, based on P-Smad1/5/8 immunostaining
(Fig. 3D). (D,E) Black and gray
arrows indicate functions for genes/pathways deduced from the current study
and those from previously published studies, respectively. Broken arrows
indicate potential interactions. The potential relationships shown here are
not intended to imply direct or linear pathways. (D) Relatively high levels of
Bmp signaling are required to specify the dorsal program, ultimately leading
to proper retinal topographic mapping. Loss of dorsal specification in
Bmpr1a-/fx;Bmpr1b-/+;Cre mice
indicates that signaling activity falls below the threshold required for DV
patterning in these mutants. For simplicity, only interactions suggested by
genetic evidence are indicated. (E) Lower levels of Bmp signaling are required
throughout the retina to maintain normal growth and to initiate neurogenesis.
Candidate downstream targets of Bmp signaling include Chx10 and
cyclin D1, as well as Fgf15. Potential regulation of Math5
expression by Mapk is, in part, postulated based on studies in
Drosophila implying Egfr-Raf-Mapk signaling in the induction of the
pro-neural gene atonal.
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