The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal axes of the eye.
ABSTRACT The vertebrate retina is highly ordered along both its dorsoventral (DV) and nasotemporal (NT) axes, and this order is topographically maintained in its axonal connections to the superior colliculus of the midbrain. Although the graded axon guidance cues that mediate the topographic mapping of retinocollicular connections are increasingly well understood, the transcriptional regulators that set the DV and NT gradients of these cues are not. We now provide genetic evidence that Vax2, a homeodomain protein expressed in the ventral retina, is one such regulator. We demonstrate that in Vax2 mutant mice, retinocollicular projections from the ventral temporal retina are dorsalized relative to wild type. Remarkably, however, this dorsalization becomes systematically less severe in progressively more nasal regions of the ventral retina. Vax2 mutants also exhibit flattened DV and NT gradients of the EphA5, EphB2, EphB3, ephrin-B1 and ephrin-B2 axon guidance cues. Together, these data identify Vax2 as a fundamental regulator of axial polarization in the mammalian retina.
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ABSTRACT: Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is therefore given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification. Developmental Dynamics, 2013. © 2013 Wiley Periodicals, Inc.Developmental Dynamics 08/2013; · 2.67 Impact Factor
- Planar Cell Polarization during Development: Advances in Developmental Biology and Biochemistry, Edited by M Mlodzik, 07/2014: pages 59-91; Elsevier Science & Technology Books.
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ABSTRACT: Background: A major step in eye morphogenesis is the transition from optic vesicle to optic cup, which occurs as a ventral groove forms along the base of the optic vesicle. A ventral gap in the eye, or coloboma, results when this groove fails to close. Extrinsic signals, such as fibroblast growth factors (Fgfs), play a critical role in the development and morphogenesis of the vertebrate eye. Whether these extrinsic signals are required throughout eye development, or within a defined critical period remains an unanswered question. Results: Here we show that an early Fgf signal, required as the eye field is first emerging, drives eye morphogenesis. In addition to triggering coloboma, inhibition of this early Fgf signal results in defects in dorsal-ventral patterning of the neural retina, particularly in the nasal retina, and development of the periocular mesenchyme (POM). These processes are unaffected by inhibition of Fgfr signaling at later time points. Conclusions: We propose that Fgfs act within an early critical period as the eye field forms to promote development of the neural retina and POM, which subsequently drive eye morphogenesis. Developmental Dynamics, 2014. © 2014 Wiley Periodicals, Inc.Developmental Dynamics 01/2014; · 2.67 Impact Factor
Although axial polarization of the retina is essential to
perception of the visual world, the molecules that specify the
retinal axes during development have not been intensively
studied. For the most part, analyses of embryonic retinal
organization and topography have instead focused on one
important read-out of retinal polarization – the cell surface
receptors and ligands that guide the precise wiring of retinal
ganglion cell (RGC) neurons to their synaptic targets in the
superior colliculus (SC) (O’Leary et al., 1999). These receptors
and ligands act in concert to organize retinocollicular
connections into a topographic map – a spatial ordering of
axonal connections in which the Cartesian coordinates of a
two-dimensional sheet of projecting RGC neurons are mapped
onto the coordinates of a second two-dimensional sheet of
target neurons in the SC. In the retinocollicular map, axons
from RGCs in the nasal retina project to and form synapses in
the caudal end of the SC, while RGCs in the temporal retina
project to targets in the rostral SC. RGCs located at
intermediate NT retinal positions project to correspondingly
intermediate caudal-rostral positions in the SC. The
dorsoventral (DV) axis of the retina is similarly mapped onto
the lateral-medial axis of the SC.
The proteins most closely tied to the topography of
retinocollicular mapping are the receptor tyrosine kinases of
the EphA family, together with their ligands, the ephrin-A
proteins (Flanagan and Vanderhaeghen, 1998; O’Leary and
Wilkinson, 1999). Two key lines of recent genetic evidence
(Frisén et al., 1998; Brown et al., 2000; Feldheim et al., 2000),
together with a large body of earlier in vitro membrane stripe
and in vivo misexpression studies, have demonstrated that a
low-nasal-to-high-temporal retinal EphA receptor gradient,
combined with a reciprocal low-rostral-to-high-caudal
collicular ephrin-A gradient, serve to order the mapping of the
NT axis of the retina onto the caudal-rostral axis of the SC.
The cell-surface molecules that mediate mapping of the
orthogonal retinocollicular axes are less well understood.
Although clear DV gradients of EphB2 and EphB3 receptor
expression have been observed in the vertebrate retina, and
medial-lateral gradients of ephrin-B ligands have been detected
in the SC (Marcus et al., 1996; Braisted et al., 1997; Holash et
al., 1997; Schulte et al., 1999), these gradients are not
reciprocally configured, as would be required for a
chemorepellent action of the ephrin-Bs, and genetic tests of the
importance of the EphB/ephrin-B signaling system to
retinocollicular mapping have yet to be reported.
Less clear still are the transcriptional control mechanisms
through which the NT and DV retinal gradients of the EphA
and EphB receptors are established during development.
Recently, two candidates for transcriptional regulators of DV
polarization of the retina – Tbx5 and Vax2 – have been
identified (Barbieri et al., 1999; Koshiba-Takeuchi et al., 2000;
Schulte et al., 1999). Vax2 (for ventral anterior homeobox 2)
Development 129, 797-804 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
The vertebrate retina is highly ordered along both its
dorsoventral (DV) and nasotemporal (NT) axes, and this
order is topographically maintained in its axonal
connections to the superior colliculus of the midbrain.
Although the graded axon guidance cues that mediate the
topographic mapping of retinocollicular connections are
increasingly well understood,
regulators that set the DV and NT gradients of these cues
are not. We now provide genetic evidence that Vax2, a
homeodomain protein expressed in the ventral retina, is
one such regulator. We demonstrate that in Vax2 mutant
mice, retinocollicular projections from the ventral
temporal retina are dorsalized relative to wild type.
Remarkably, however, this
systematically less severe in progressively more nasal
regions of the ventral retina. Vax2 mutants also exhibit
flattened DV and NT gradients of the EphA5, EphB2,
EphB3, ephrin-B1 and ephrin-B2 axon guidance cues.
Together, these data identify Vax2 as a fundamental
regulator of axial polarization in the mammalian retina.
Key words: Retina, Homeobox, Vax genes, Emx genes, Dorsoventral
axis, Axon guidance, Mouse
The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal
axes of the eye
Stina H. Mui1,2, Robert Hindges1, Dennis D. M. O’Leary1, Greg Lemke1,* and Stefano Bertuzzi1,†
1Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037 USA
2Department of Neurosciences, University of California San Diego, La Jolla, CA 92093 USA
*Author for correspondence (e-mail: email@example.com)
†Present address: Telethon Foundation at CNR Istituto Tecnologie Biomediche, 20090 Segrate (Milan), Italy
Accepted 6 November 2001
is one of two vertebrate-specific homeobox genes that are
structurally related to, and that have almost certainly evolved
from the duplication of, the vertebrate Emx genes (Barbieri et
al., 1999; Hallonet et al., 1998; Ohsaki et al., 1999). Loss-of-
function experiments for Vax1 have demonstrated that its
product plays essential roles in axon guidance and major tract
formation in the developing forebrain (Bertuzzi et al., 1999;
Hallonet et al., 1999). Vax2, which carries a homeodomain
identical to that of Vax1, is a candidate regulator of the retinal
DV axis for two reasons. First, it is steeply graded in its
expression along this axis in chick and frog embryos, with
highest expression ventrally. And second, dominant gain-of-
function studies in these embryos have shown that, when
misexpressed in the dorsal retina, Vax2 is capable of
ventralizing this tissue, as assessed by (1) the altered
expression of DV marker genes, both putative guidance cues
such as EphB2/B3 and putative transcriptional regulators such
as Tbx5, and (2) the altered projection of RGC axons to the
midbrain. These observations have been interpreted as
indicating that Vax2 may function as a global ‘ventralizing’
regulator of the developing eye. In this report, we describe the
generation of Vax2–/–mice, and the use of these mutants to
perform loss-of-function tests of this hypothesis.
MATERIALS AND METHODS
The mouse Vax2 gene was cloned from a strain 129/sv genomic library
and was inactivated by deletion/replacement (see Fig. 1G). Vax2–/–
mice were generated by standard procedures (Tybulewicz et al., 1991).
Electroporated W95 embryonic stem (ES) cell clones were doubly
selected with G418 and FIAU, and were screened for homologous
recombination by Southern blot, using the 230 bp 3′-external probe
indicated in Fig. 1. Four positive ES cell clones were microinjected
into C57/Bl6 blastocysts to generate chimeric mice, which were then
mated with C57/Bl6 to produce heterozygous knockouts, all
according to standard procedures (Capecchi, 1989). Although most
Vax2–/–mice did not exhibit overt postnatal phenotypes, ~7% of
homozygotes were abnormal, in that they either were unusually small
in size, exhibited a trembling phenotype or displayed small eyes.
In situ hybridization with 33P-radiolabeled or digoxigenin-labeled
riboprobes were performed as described previously (Braisted et al.,
1997; Zhadanov et al., 1995). Dissected mouse embryos were
anesthetized and immersion fixed in 4% paraformaldehyde/PBS
overnight at 4°C, followed by overnight infiltration with 20% sucrose.
Cryoprotected tissues were then embedded in OCT medium (Miles).
Hybridization was performed with 20 µm coronal and transverse
sections of retina at birth (P0), and with embryos at E9.5, E11.5 and
In vivo anterograde labeling was performed between postnatal day 7
(P7) and P9 by focal injection of 1,1′-dioctadecyl-3,3,3′3′-
tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes;
8% w/v in N,N-dimethylformamide) into the left eye, as described
previously (Brown et al., 2000; Simon and O’Leary, 1992). Mice were
sacrificed 1 day later, and termination zone (TZ) number and location
in the contralateral superior colliculi were analyzed using a Zeiss
LSM510 confocal microscope. The size and location of focal retinal
DiI injection sites were identified post hoc.
Intraocular injection of fluorescently tagged cholera toxin B-
subunit tracers (CTB) into one eye allowed for visualization of both
ipsilateral and contralateral retinal projections. For anterograde
tracing of the RGC axons, CTB conjugated to a fluorescein (FITC)
fluorophore (List Biological Laboratories, Campbell, CA) was made
in a 0.2% solution in 1% DMSO. Whole eye fill injections were
carried out at P8 and the animals were perfused with 4%
paraformaldehyde 24 hours later. Brains were cut coronally in 200 µm
sections and subsequent imaging was performed using a confocal
Dual gradients of Vax2 expression in the mouse
Although the Vax2 homeodomain is identical to that of Vax1
(Barbieri et al., 1999; Schulte et al., 1999), the two Vax genes
nonetheless exhibit distinct and largely complementary
patterns of expression during embryogenesis in the mouse. At
embryonic day 10.5 (E10.5) and thereafter, Vax1 mRNA is
expressed in the ventral diencephalon and telencephalon, and
in the optic stalk and disk, but is not prominently expressed in
the developing neural retina (Bertuzzi et al., 1999; Hallonet et
al., 1999). By contrast, the expression of mouse Vax2 mRNA
is largely confined to the ventral half of the developing eye
itself (Fig. 1A). These distinct embryonic expression domains
in the mouse contrast with the situation in the chick, where a
single gene (VAX) exhibits a hybrid Vax1/Vax2-like expression
profile throughout the ventral diencephalon, optic stalk, and
ventral retina (Schulte et al., 1999). At E11.5, mouse Vax2
mRNA is expressed in all cells of the ventral neural retina (Fig.
1B), but by birth becomes restricted to ventral RGCs (Fig. 1E).
In addition to the previously reported high-ventral-to-low-
dorsal retinal gradient of Vax2 mRNA (Fig. 1B,E), we have
observed a shallower Vax2 gradient along the NT axis, with
highest expression nasally (Fig. 1B-D,F). This dually polarized
expression is evident throughout the neural retina at E14 (Fig.
1C,D), when RGC axons first reach the SC, and persists at least
until birth in the mouse, when the DV patterning of RGC
projections is well under way. The double Vax2 retinal gradient
in the mouse is similar in orientation and configuration to the
recently described dual retinal expression gradient of the bone
morphogenetic protein 4 (BMP4) antagonist ventroptin in the
chick (Sakuta et al., 2001).
Inactivation of the mouse Vax2 gene
In order to test the hypothesis that Vax2 may specify positional
identity in the retina along the DV axis (Barbieri et al., 1999;
Schulte et al., 1999), we generated Vax2 mutant mice (Fig. 1G-
I). We replaced exon 2 of the mouse Vax2 gene, which encodes
the first two essential α helices of the Vax2 homeodomain, with
a G418 resistance (PGK-neo) cassette. In addition to deleting
exon 2, this mutation introduces a frame shift that eliminates
all Vax2-coding sequence downstream of exon 2 (see Fig. 1G,
Materials and Methods).
Retinocollicular mapping in the Vax2 mutants
We first analyzed the projection of RGC axons from the retina
to the SC in the Vax2 mutants. Normally, the DV and NT axes
of the retina are precisely mapped onto the correspondingly
orthogonal lateral-medial and caudal-rostral axes of the SC
(Brown et al., 2000). If Vax2 is required for ventral patterning
S. H. Mui and others
799 Vax2 patterns eye axes
of the retina, then ventral RGCs that lack Vax2
should be dorsalized in terms of their projection
to the SC; that is, they should project to lateral
rather than to medial SC. In mice, the topography
of the retinocollicular map is mature by postnatal
day 7 (P7) (O’Leary et al., 1986). We found that
most of the Vax2–/–mice (94 out of 101) were
healthy and superficially normal for many months
after birth; in marked contrast to Vax1 mutants
(Bertuzzi et al., 1999), no postnatal colobomata
were observed. We therefore labeled discrete loci
of RGCs and their projecting axons across the full
extent of the NT and DV axes of the mutant retina
at P7-9. We performed focal injections of the
lipophilic axon tracer DiI (Fig. 2), and then
analyzed labeled projections to the contralateral
SC 1 day later (see Materials and Methods). In
total, we analyzed 40 injections (from 40 mutant
mice). The Vax2 mutant retinae that we selected
for study were histologically indistinguishable
from wild type in terms of retinal lamination,
cellular density, organization of plexiform layers
and closure of the optic disk (data not shown).
Given that Vax2 is not expressed in the extreme
dorsal retina, axon projections from dorsal RGCs
should not be altered in the Vax2 mutants. This
was the case. The dorsal RGC axons of mutant
mice (n=8 dorsal injections, at varying NT
positions, in 8 mice) entered the SC at its rostral
lateral edge and formed tight termination zones
(TZs) at expected locations in the lateral SC (Fig.
2S, and data not shown). In marked contrast, axon
projections from most Vax2–/– ventral RGCs did
not, with RGC axons from the ventral temporal
retina being the most strongly affected. These
ventral temporal axons entered the SC at the
lateral rostral edge, and always formed single,
well-circumscribed TZs in the lateral rostral SC
(n=10/10) (Fig. 2A, lower panel, Fig. 2D-F). As
both wild-type and Vax2+/–ventral temporal
axons project to medial rostral SC (Fig. 2A,
middle panel), the projection pattern of these
Vax2–/–RGCs represents a complete retinal
dorsalization of their mapping behavior. This
dorsalization was robust and fully penetrant (Fig.
2D-F), and was reflected not only in the position
of the TZs, but also in the site at which RGC
axons entered the rostral colliculus (compare
middle and lower panels in Fig. 2A).
Unexpectedly, however, the dorsalization of
RGC projections became progressively less
severe as DiI injections were moved to
increasingly nasal regions of the ventral retina. At
80-85% of the retinal NT axis (where extreme
temporal=100%), ventral RGC TZs remained
aberrantly lateralized in their SC projection, but more than one
lateral TZ was typically observed (Fig. 2G,H). As injections
were moved to 60-80% of the NT axis, the size of ectopic
lateralized TZs became progressively smaller, and an
appropriate TZ appeared very near the expected medial
position (Fig. 2I-K). For mid-ventral injections into the Vax2
mutant retinae (30-60% of the NT axis), collicular TZs
typically appeared near the expected medial location,
occasionally as single, well-formed TZs (Fig. 2M). When
ectopic lateral TZs were observed, they were most frequently
small (Fig. 2B, lower panel and Fig. 2N-R). Finally, ventral DiI
injections into the most nasal regions of the Vax2 mutant retina
Fig. 1. Vax2 mRNA expression in the mouse and inactivation of the mouse Vax2
gene. (A) Whole-mount in situ hybridization for Vax2 mRNA in an E9 mouse
embryo. (B) High-power view of an E11.5 mouse eye, illustrating the pronounced
dorsal-ventral (DV) Vax2 gradient. D, V, N, and T indicate dorsal, ventral, nasal and
temporal poles of the retina. (C,D) Front (C) and back (D) views of a whole-mount
in situ hybridization for Vax2 mRNA (purple reaction product) in a dissected mouse
retina at E14. Arrowhead denotes a ventral cut made before the eye was dissected
from the embryo. (E) In situ hybridization of a coronal section through a wild-type
P0 mouse eye, demonstrating high expression of Vax2 mRNA in the retinal ganglion
cells (rgc) of the ventral (V) but not the dorsal (D) retina. (F) In situ hybridization of
a transverse section through a wild-type P0 mouse eye, demonstrating slightly
higher expression of Vax2 mRNA in the retinal ganglion cells (rgc) of the nasal (N)
than the temporal (T) retina. (G) Targeting construct for inactivation of the mouse
Vax2 gene, and structure of the inactivated allele after homologous recombination in
mouse embryonic stem cells. A BamHI/EcoRV genomic fragment containing exon 2
of the Vax2 gene was replaced by a PGK-neo cassette (see Materials and Methods).
Exon 2 encodes amino acid residues 83-145; these residues include the first two α
helices of the Vax2 homeodomain, which are essential to the function of all known
homeodomain transcription factors. The BamHI/EcoRV deletion also introduces a
shift in the Vax2 reading frame. (H) Southern blot of XbaI and SalI-digested
genomic DNA from wild-type (+/+) and heterozygous (+/–) ES cell clones probed
with the external 3′ probe indicated in G and described in Materials and Methods.
(I) RT-PCR analysis of mRNA isolated from wild-type (+/+) and Vax2 mutant (–/–)
retinae at P0. The primers for the PCR reaction are located in exons 1 and 3, and
correspond to nucleotides 127-148 and 586-606 of the mouse Vax2 cDNA sequence
(Genbank Accession Number, Y17792). The size of the –/– fragment is consistent
with the clean excision of exon 2, which was confirmed by DNA sequence analysis
of the 468 and 261bp bands, and by the loss of Vax2 immunoreactivity in the
homozygous mutant retina (data not shown). Scale bar: 0.1mm in E,F.
(0-30% of the retinal NT axis) almost always yielded single
tight TZs at the expected wild-type location in the medial SC
(Fig. 2C, Fig. 2T-W). Innervation space in the rostral medial
quadrant of the Vax2–/–SC vacated by lateralized ventral
temporal RGC axons appeared to be occupied by TZs from
RGCs whose axons would normally occupy extreme medial
locations near the midpoint of the RC axis (Fig. 2L). Thus, the
Vax2–/–axon projection phenotype progressed from extremely
strong and fully penetrant in the ventral temporal retina (Fig.
2A,D-F) to almost non-existent in the ventral nasal retina (Fig.
2C,U-W). Note that this NT phenotypic progression is
the inverse of the shallow gradient of Vax2 mRNA (see
Maintenance of ipsilateral projections
At maturity, the vast majority (>95%) of RGC axons in the
mouse cross at the optic chiasm and innervate the contralateral
SC; the rare ipsilateral projections originate primarily in the
ventral temporal quadrant of the retina (Sretavan and Kruger,
1998). Although ventral temporal RGC projections are those
that are most strongly perturbed in the contralateral SCs of the
Vax2 mutants, we nonetheless consistently detected ipsilateral
S. H. Mui and others
Fig. 2. Aberrant projections of retinal ganglion cell (RGC) axons from
the retina to the superior colliculus (SC) in Vax2 mutants, as assessed by
anterograde axonal tracing with DiI. (A) A focal DiI injection into the
ventral temporal (VT) quadrant of the retina (indicated by the circle on
the upper panel retinal flatmount schematic) labels RGC axons that
terminate at the medial-rostral (MR) border of the SC in wild-type and
heterozygous mice [+/–, middle panel, arrow indicates termination zone
(TZ)], but at the rostral-lateral border of the SC in the mutant (–/–, lower
panel, arrow). Note that for all anterograde labeling we analyzed, the
projections of heterozygous Vax2+/–axons were indistinguishable from
the previously described projections of wild-type RGCs. (B) A mid-
ventral injection (split circle on schematic) labels RGC axons that terminate near the medial border of the SC at the midpoint of the collicular
RC axis in both heterozygous (+/–, middle panel, arrow) and mutant mice (–/–, lower panel, arrow). Arrowheads indicate additional multiple
ectopic TZs in the mutant, some of which are lateralized. (C) A focal injection into the ventral nasal quadrant of the retina (circle, upper panel)
labels RGC axons that terminate in the medial-caudal SC in both heterozygous (+/–, middle panel) and Vax2 mutant mice (–/–, lower panel).
(D-W) SC diagrams depicting expected (squares) and observed collicular TZs (gray) from focal DiI injections into the ventral retinae of Vax2
mutants, illustrating the progressive change in misprojection phenotype from extremely strong for ventral temporal injections (D-F) to
extremely weak for ventral nasal injections (U-W). The position of the expected TZ in the SC is plotted from the observed position of the
retinal injection site, assuming a linear map from extreme ventral retina=extreme medial SC to extreme dorsal retina=extreme lateral SC.
Squares and gray shapes are scaled to indicate relative sizes of retinal injection sites and collicular TZs, respectively. With the exception of S,
all panels illustrate results from ventral retinal injection between 60-85% of the DV axis, with extreme dorsal defined as 0%. c, caudal; l,
lateral; m, medial; r, rostral.
801 Vax2 patterns eye axes
RGC projections after full eye fills with fluorescent
anterograde axonal tracers in these mice (Fig. 3; n=5). Indeed,
in some mice, these ipsilateral projections appeared to be
Flattened DV gradients of axon guidance cues
The aberrant RGC projections seen in the Vax2 mutants suggest
that this transcription factor normally patterns the mouse retina
beginning around E10, and thereby directly or indirectly
controls expression of later position-dependent axon guidance
cues. Vax2 misexpression in the dorsal chick and frog retina
results in (1) the upregulated expression of mRNAs encoding
the EphB2 and EphB3 receptor tyrosine kinases, which are
normally ventrally restricted, and (2) the downregulated
expression of mRNAs encoding their ligands ephrin-B1 and
ephrin-B2, which are normally dorsally restricted (Barbieri et
al., 1999; Schulte et al., 1999). Similarly, mRNA encoding a
dorsally restricted transcription factor and presumed DV
regulator – Tbx5 – is downregulated by these gain-of-function
manipulations. Consistent with a subset of these earlier
observations, we found that ventral expression of both the
Ephb2 and Ephb3 mRNAs was lost in the Vax2–/–ventral retina
(Fig. 4A and data not shown), and that conversely, ventral
expression of the ephrin-B1 and ephrin-B2 mRNAs (Efnb1 and
Efnb2 – Mouse Genome Informatics) was acquired (Fig. 4B
and data not shown). In addition to these DV alterations, and
consistent with our detection of a shallow NT Vax2 gradient,
we also observed a partial reordering of the retinal NT axis in
the ventral retina of the Vax2–/–mutants. For example, Epha5
mRNA, which is normally distributed in a low-nasal-to-high-
temporal gradient throughout the DV axis (Brown et al., 2000),
was upregulated in the ventral nasal mutant retina (Fig. 4C).
Thus, each of the above changes leads to flattened or abolished
gradients of guidance cue mRNAs that are normally graded
along both the DV and NT axes (compare left and right panels
in Fig. 4A-C). This global regulation of multiple guidance cues
is apparently crucial, as preliminary analysis of mice doubly
mutant for the Ephb2 and Ephb3 genes indicates that medial-
lateral RGC axon targeting defects in these double EphB
mutants are much less pronounced than those detailed above
for the Vax2 single mutants (R. H., T. McLaughlin and D. D.
M. O., unpublished).
Maintained DV gradients of transcriptional
While Vax2 orchestrates the expression of several genes that
encode graded axon guidance cues, the gradients of
transcription factors that have been hypothesized to define
retinal axial polarity, and thereby set up the guidance cue
gradients, remained unchanged in the Vax2 mutants. Notably,
the expression of Pax2, an early embryonic marker of the
ventral retina (Koshiba-Takeuchi et al., 2000; Schulte et al.,
1999) remained graded, low-dorsal-to-high-ventral, in early
Vax2 mutant embryos (data not shown). Similarly, the inverse
high-dorsal-to-low-ventral gradient of mRNA encoding the T
box transcription factor Tbx5, which is the earliest known
dorsal retinal marker (Koshiba-Takeuchi et al., 2000), was,
unlike ephrin-B1 and -B2, unchanged in the Vax2 mutants at
E10.5 and P0 (Fig. 4D, and data not shown). Although gain-
of-function Vax2 misexpression experiments have suggested
that Tbx5 is normally repressed by Vax2 (Schulte et al., 1999),
our results demonstrate that this is not the case: loss of Vax2
in the ventral retina does not lead to Tbx5 upregulation. As
Tbx5 misexpression experiments have similarly suggested that
Vax2 may be repressed by Tbx5 (Koshiba-Takeuchi et al.,
2000), analysis of retinal polarity in Tbx5 loss-of-function
mutants will be required in order to clarify the relationship
between these transcription factors. Nonetheless, our results
indicate that the highly polarized expression of Tbx5 does not
require Vax2. Similarly, the homeodomain protein Six6 and the
winged-helix transcription factor Bf1 (Foxg1 – Mouse Genome
Informatics) (Huh et al., 1999; Lopez-Rios et al., 1999), which
Fig. 3. Maintained ipsilateral projections in the Vax2 mutants. Whole single eye injections with fluorescent cholera toxin B subunit label
abundant contralateral (contra) and rare ipsilateral (ipsi) RGC projections in both Vax2+/–(upper panels) and Vax2–/– (lower panels) mice at P7.
Arrowheads indicate ipsilateral termination sites of RGCs in successive rostral to caudal serial coronal sections, spaced at 200 µm, which
traverse the thalamus into the SC. The ventral midline is indicated by the vertical lines.
are also hypothesized regulators of retinal axial polarity, and
which at E9.5 exhibit expression in the ventral nasal (Bf1) and
ventral temporal (Six6) mouse retina, respectively (Fig. 4E,F),
were unchanged in their expression domains in the Vax2
mutants (Fig. 4G, and data not shown). These domains are
dynamic for both Bf1 and Six6 from E9.5 to birth: Bf1 is first
expressed in the ventral nasal retina (Fig. 4E) and by E12.5
moves to the dorsal nasal retina; and Six6 is first detected in
the ventral temporal retina (Fig. 4F), but later occupies the
entirety of the retina. These dynamic expression domains were
unaffected in the Vax2 mutants, as assessed by in situ
hybridization at E10.5, E12.5, E14.5, E16.5 and P0 (Fig. 4G,
and data not shown).
Our loss-of-function analyses definitively demonstrate that
Vax2 controls DV polarization of the mouse retina. This
transcription factor directly or indirectly activates expression
of ventral axon guidance cues such as EphB2 and EphB3, and
at the same time represses dorsal cues such as ephrin-B1 and
ephrin-B2. In the temporal regions of the ventral retina, the loss
of Vax2 results in RGC projections to the contralateral SC
that are completely dorsalized relative to their wild-type
These results notwithstanding,
demonstrate that the ventralizing influence of Vax2 is in two
respects incomplete. First, although Vax2 expression normally
extends across the entirety of the ventral retina, the
dorsalization of RGC projections in the Vax2 mutant retina
does not. As detailed above (Fig. 2), dorsalization is robust
only in the extreme ventral temporal retina, and becomes
progressively less severe in progressively more nasal regions
of the retina. In the extreme ventral nasal retina, mutant RGC
projections are indistinguishable from wild type. And second,
some of the phenotypes seen upon loss of Vax2 are not
consistent with those reported for Vax2 gain-of-function
studies. Most notably, loss of Vax2 does not lead to the
activation of Tbx5 expression in the ventral retina, or to any
other obvious perturbation in the high-dorsal-to-low-ventral
gradient of Tbx5 expression. These latter observations suggest
that transcriptional specifiers of vertebrate retinal polarity, like
many transcriptional specifiers of retinal identity (Marquardt et
al., 2001), operate independently.
The surprising finding that the axonal targeting errors of
ventral temporal RGCs are much more severe than those of
ventral nasal RGCs suggests that either: (1) Vax2 interacts with
our analyses also
S. H. Mui and others
Fig. 4. Flattened gradients of axon
guidance cues and maintained gradients
of transcription factors in the retinae of
Vax2 mutants at birth. (A) Ephb2 mRNA
(visualized as pink/red silver grains) is
expressed in a high ventral (V)/low
dorsal (D) gradient in both the wild-type
and heterozygous retina (+/–, lower and
upper left panels, respectively), and is
lost in the ventral retina of the mutants
(–/–). The positions of the retinal
ganglion cell and pigment epithelial
layers are denoted by filled and open
arrowheads, respectively, in the upper
left panel. (The intense signal seen in the
pigment epithelial layer of nearly all
panels is an artifact caused by refraction
of pigment when viewed with dark field
optics. The image used for C was, by
chance, generated from an unpigmented
mutant.) Essentially identical results
were obtained for mRNA encoding the
closely related and similarly graded
receptor EphB3. (B) mRNA encoding
ephrin-B1, a ligand for EphB2/B3, is
expressed in a high dorsal (D)/low
ventral (V) gradient, the inverse of
EphB2/B3, in the heterozygous retina
(+/–, left panels), and this mRNA is
upregulated in the ventral retina of the
mutants (–/–, right panels). Essentially identical results were obtained for mRNA encoding the closely related and similarly graded ligand ephrin-
B2. (C) Epha5 mRNA is expressed in a high temporal (T)/low nasal (N) gradient in the heterozygous retina (+/–, left panels), and is upregulated
in the ventral nasal mutant retina of the mutant (–/–, right panels). (D) mRNA encoding the transcription factor Tbx5 exhibits a pronounced high-
dorsal-to-low-ventral gradient of expression in wild-type P0 mouse retinae (Koshiba-Takeuchi et al., 2000), and this gradient is not changed in
the Vax2 mutants (–/–). (E) Expression of Bf1 mRNA (dark purple reaction product) at E9.5 in wild type is most pronounced in the ventral nasal
quadrant (arrowhead) of the optic vesicle (circled). Anterior is towards the left. (F) Expression of Six6 mRNA at E9.5 in wild type is most
pronounced in the ventral temporal quadrant (arrowhead) of the optic vesicle (circled). Anterior is towards the left. (G) Radioactive in situ
hybridization to transverse sections of heterozygous (+/–) and mutant (–/–) retinae at E16.5 demonstrate unaltered expression of Bf1 mRNA,
which remains preferentially expressed in the nasal (N) as opposed to temporal (T) side of the eye. Scale bar: 0.1 mm.
803Vax2 patterns eye axes
one or more additional transcriptional regulators that are
similarly graded along the DV and NT axes, such that a
‘limiting concentration effect’ of these nuclear proteins is
detected in the mutants only in the retinal quadrant where their
aggregate level is normally lowest (Fig. 5A); or (2) the Vax2
gene, which is steeply graded along the retinal DV axis,
normally interacts genetically with one or more genes that are
steeply graded along the NT axis (Fig. 5B). With regard to the
second possibility, other homeodomain transcription factor
genes, such as SOHO1 and GH6 in the chick (Schulte and
Cepko, 2000), have been shown to be graded along the retinal
NT axis, and have been hypothesized to control the expression
of NT guidance cues such as EphA receptors and their ephrin-
A ligands. The homologs of SOHO1 and GH6 remain to be
analyzed in the mouse, but the transcription factor Bf1 is
specifically expressed, from E8.5-E9.5, in the mouse ventral
nasal retina (Fig. 4E). Although the EphA/ephrin-A guidance
cues have been analyzed almost exclusively in terms of RGC
mapping along the rostral-caudal axis of the SC (Brown et al.,
2000; Feldheim et al., 2000), it is interesting to note that
medial-lateral mapping anomalies have been consistently
observed in ephrin-A2/A5 double mutants (Feldheim et al.,
2000). If, as schematized in Fig. 5B, Vax2 normally acts in
concert with one or more transcription factors that are graded
along the NT axis, then RGCs in the ventral temporal retina
would again be those most sensitive to the loss of Vax2. Direct
tests of these and related models must await the generation and
analysis of a set of single and compound mutants of Vax2 with
Bf1, SOHO1, GH6, ephrin-A2 and ephrin-A5, among others.
Together, our results identify Vax2 as an essential specifier
of both DV and NT axial polarity in the developing mammalian
eye. They demonstrate that the steeply graded expression of
Vax2 that first appears near the midpoint of mouse
embryogenesis is essential to the ventral specification of the
retina, most prominently in its ventral temporal quadrant.
Importantly, they further demonstrate that this specification is
independent of more widely expressed transcription factors
that have also been thought to act during axial determination
of the eye, and suggest that these regulators normally act in
concert with Vax2.
This work was supported by grants from the NIH and the Italian
Telethon Foundation (G. L., D. D. M. O. and S. B.), by postdoctoral
fellowships from the Italian Telethon Foundation (S. B.) and the Swiss
National Science Foundation (R. H.), and by the Medical Scientist
Training Program at UCSD (S. M.). We thank Qingxian Lu and Todd
McLaughlin for helpful advice and discussion, Arthur Brown and
Todd McLaughlin for in situ hybridization probes, and Patrick
Burrola, Darcie Baynes, Dario Strina and Arjay Clemente for
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Fig. 5. Two models for the concerted
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transcriptional regulators along the nasal-
temporal (NT) axis of the ventral retina.
(A) Ventral regulators are normally expressed
at higher levels in the nasal than the temporal
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that are graded along the NT axis (middle discs), to form a composite wild-type map of axon guidance regulators (right discs). With the loss of
Vax2, the map reverts to that of the NT genes alone, which again most severely affects ventral temporal RGCs. See text for details.
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