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First published online 19 July 2006
doi: 10.1242/dev.02485
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Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA.
* Author for correspondence (e-mail: monte{at}uoneuro.uoregon.edu)
Accepted 12 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Axin, Forebrain, Lhx5, LIM-homeobox domain factor, masterblind, Secreted Wnt antagonist, Sfrp, Wnt signaling, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Later in development, local interactions within
the forebrain pattern and regulate the differentiation and growth of rostral
neural precursor cells to give rise to various forebrain structures
(Munoz-Sanjuan and Brivanlou,
2002; Wilson and Edlund,
2001; Wilson and Houart,
2004). Functional studies of these extrinsic signals, produced by
the mesendoderm and its derivatives, and of their inhibitors have contributed
significantly to our understanding of forebrain development
(Harland and Gerhart,
1997).
The functional roles that the rostral neuroectoderm itself may play in
formation of the forebrain are less well understood. Several studies have
suggested that rostral neuroectoderm factors antagonize ventralizing and
caudalizing signals. In the Xenopus blastula organizer, for example,
cells required for brain formation express the Bmp signaling inhibitors
Chordin and Noggin (Kuroda et al.,
2004). In zebrafish, Tlc, a secreted frizzled-related protein
expressed at the rostral margin of the neural plate, antagonizes Wnt signaling
(Houart et al., 2002). In
mouse, Six3 in the rostral ectoderm directly represses Wnt1
expression (Lagutin et al.,
2003). In Xenopus, Shisa functions in the endoplasmic
reticulum to antagonize Wnt and Fgf signaling by preventing the maturation of
Wnt receptors and Fgf receptors (Yamamoto
et al., 2005).
How are these antagonists of caudalizing signals regulated? We discovered
that Lhx5, a transcription factor expressed by rostral ectoderm, is a
regulator of Wnt antagonists. Lhx5 belongs to a family of LIM-homeodomain
transcription factors, and contains two LIM protein interaction domains and a
homeodomain (Hobert and Westphal,
2000; Retaux and Bachy,
2002; Toyama et al.,
1995). Previous studies suggested that Lhx5 regulates the
differential adhesion of early ectodermal cells in Xenopus
(Houston and Wylie, 2003), and
morphogenesis and cell proliferation in the mouse hippocampus
(Zhao et al., 1999). We show
that Lhx5 gain of function in zebrafish inhibits Wnt signaling, whereas
inhibition of Lhx5 results in ectopic activation of Wnt signaling and
forebrain defects. Lhx5 regulates expression of the secreted Wnt antagonists
Sfrp1a and Sfrp5, and Sfrp gene gain of function can rescue the forebrain
developmental defects caused by the inhibition of Lhx5 function. We propose
that Lhx5 is an intrinsic factor required for forebrain development because it
inhibits Wnt signaling by regulating the local expression of secreted Wnt
antagonists.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
masterblind line (mbltm213) was provided by the
Zebrafish International Resource Center (Eugene, OR). RFLP genotyping of
mbltm213 embryos was carried out by PCR using the primers
listed below. The mbltm213 mutation abolishes a
BpmI site in the amplified fragment. mbl-forward, 5'-GAGGTGTTTTCTCCACAGCATC-3'; and
mbl-reverse, 5'-TACACCAGGAAATTCATCCAGTC-3'.
Whole-mount in situ hybridization
Embryos were staged as described
(Kimmel et al., 1995). The
sphere-dome transition was used as the reference time point to stage blastula
and early gastrula embryos.
Whole-mount in situ hybridization was performed as described
(Thisse et al., 1993;
Whitlock and Westerfield,
2000). The clones used in this study have been previously
described: lhx5 (Toyama et al.,
1995), ptc1 (Concordet
et al., 1996), emx3
(Morita et al., 1995),
pax6a (Puschel et al.,
1992) and pax2a
(Krauss et al., 1991). The
clone used for the synthesis of the six3b probe was obtained from the
Zebrafish International Resource Center.
Cloning and phylogeny
Zebrafish Sfrp genes were amplified by RT-PCR based on the sequences
obtained from a BLAST search of the zebrafish genome
(http://www.sanger.ac.uk/cgi-bin/blast/submitblast/d_rerio
and
http://www.ensembl.org/Danio_rerio/).
Multiple sequence alignment was performed with ClustalX using BLOSUM
protein weight matrix (Thompson et al.,
1994). The phylogenetic tree was reconstructed by a Bayesian
method with MrBayes
(http://mrbayes.csit.fsu.edu/index.php),
using mixed model and Gamma distributed rates. The phylogram was drawn using
TreeView (Page, 1996). Gene
name assignments were corroborated by synteny analyses (details available upon
request) and approved by the Zebrafish Nomenclature Committee
(http://zfin.org/zf_info/nomen.html).
Sequences for sfrp1a and sfrp5
(Hirate et al., 2001) are
essentially identical to two existing mRNA sequences in GenBank (NM_205585 and
NM_131858). Zebrafish axin2
(Shimizu et al., 2000) and
wnt8a orf1 (Lekven et al.,
2001) were amplified by RT-PCR based on the GenBank sequence
NM_131561 and NM_130946, respectively.
Synthetic mRNA and morpholinos
PCR-amplified regions containing lhx5 (-295 to 1251, using the
first nucleotide of the start codon as the reference), lhx5trunc
(-295 to 799, encodes amino acid residues 1-266, wnt8a orf1 (-15 to
1094), sfrp1a (-152 to 891) and sfrp5 (-152 to 1605) were
inserted into the pCS2 vector. The Drosophila Engrailed repressor
domain (residues 2-297) was fused to Lhx5 at the C terminus of the homeodomain
by subcloning a T4 polymerase-treated SphI-XbaI fragment of
pCS2-EnR into the BbsI-XbaI cut and T4 polymerase-treated
pCS2-lhx5 construct. The dosages for mRNA injection were 200 pg of
lhx5, 200 pg of lhx5trunc, 20 pg of wnt8a orf1, 200
pg of lhx5-en, 300 pg of sfrp1a and 100 pg of sfrp5
mRNA per embryo.
Morpholino (Gene Tools) lhx5-MO targets the translation start site and lhx5-e3i3 targets the third intron splice donor site. The dosages injected were 2 ng or 4 ng of lhx5-MO, and 5 ng of lhx5-e3i3 per embryo.
lhx5-MO, 5'-GTGCACCATCATTCCGCCCTGGAGG-3'; and
lhx5-e3i3, 5'-GTGCGTTGTTCTCACCTGAATCACC-3'.
Primers used in RT-PCR shown in Fig. S1 in the supplementary material are listed below:
lhx5-exon 3-forward, 5'-GATCAAATCCAGGACGACACGAAG-3';
lhx5-exon 5-reverse, 5'-GAACCCGAGCTGAGAAGATAAGG-3';
odc1-forward, 5'-CGAACCCTGATGTACTACGTGAATG-3'; and
odc1-reverse, 5'-CAGGCTGCACTGCTCCACAATG-3'.
Cell transplantation
Transplantations were carried out as described
(Dutta et al., 2005). Cells
were taken from the animal poles of midblastula stage donor embryos and
transplanted to the animal poles of late blastula stage hosts. Donor embryos
were injected with lysine fixable fluorescein dextran for tracing (Molecular
Probes), and donor cells within host embryos were revealed in red by
antifluorescein antibody labeling.
Chromatin immunoprecipitation
Chromatin immunoprecipitation experiments were carried out according to a
standard protocol (Oberley and Farnham,
2003). In brief, embryos were injected with lhx5-GFP mRNA
at the one-cell stage and fixed with 1% formaldehyde at 80% epiboly.
Uninjected embryos were treated in parallel and served as controls.
Cross-linked chromatin samples were fragmented to an average length of 1 kb by
sonication. Rabbit anti-GFP antibody (1 µl) was added to each chromatin
sample containing approximately 107 copies of haploid genome. DNA
samples from immunoprecipitated chromatins were purified and then analyzed by
PCR (primers listed below). Similar results were obtained from three
independent injections and chromatin preparations.
sfrp1a promoter-forward, 5'-GTGTGGAACTCTCCAACAGGAG-3';
sfrp1a promoter-reverse, 5'-TGGCTGTGAGTGGAAAAGTGAC-3';
aldoaa-forward, 5'-GCAGACATTTGAGAGATGAAAGG-3';
aldoaa-reverse, 5'-CATGCTGCTACATGCACAAACTG-3';
bactin2-forward, 5'-TCGATTACCGATTAAACGTGGAC-3'; and
bactin2-reverse, 5'-CGCACCAATACCACTCAACAAG-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
first detect lhx5 transcripts on the presumptive dorsal side of
midblastula stage embryos at 40% epiboly. By the onset of gastrulation,
lhx5 mRNA is distributed in a dorsal to ventral gradient in the
rostral ectoderm (Fig. 1A,B).
During gastrulation, lhx5 expression is restricted to the presumptive
forebrain (Fig. 1C,D). Later,
lhx5 expression is further restricted to subdomains in the
telencephalon, diencephalon, tegmentum, hindbrain and spinal cord
(Toyama et al., 1995). We find that excess Lhx5 activity expands the size of the forebrain. Injection of lhx5 mRNA into one-cell stage embryos results in enlarged rostral head structures by mid-somitogenesis stages when compared with uninjected control embryos (Fig. 1E,F; 40%, n=108; see also Fig. S2 in the supplementary material). Expansion of presumptive forebrain in the injected embryos is also indicated by an enlarged pax6a expression domain in the rostral neural plate by the end of the gastrulation (Fig. 1G,H; 57%, n=65). To examine the relative expansion of different brain regions, we labeled lhx5 mRNA-injected embryos and uninjected controls with the presumptive telencephalon marker emx3, the mid-hindbrain boundary marker pax2a, and the hindbrain marker egr2b (Fig. 1I-K, 70%, n=30; Fig. 1L-N, 53%, n=32). These markers indicate that the presumptive forebrain is expanded, whereas the midbrain and hindbrain are unaffected.
Lhx5 activity is required for forebrain development. To inhibit Lhx5 function, we used two approaches: overexpression of a dominant repressor construct that produces a dominant interfering protein and injection of antisense morpholino oligonucleotides that block Lhx5 protein synthesis. We generated the dominant interfering construct, lhx5-en, by replacing the Lhx5 transcriptional activation domain with the Drosophila Engrailed repressor domain. Injection of lhx5-en mRNA results in embryos that lack the most rostral part of the head; posterior head structures and other parts of the embryo are unaffected (Fig. 1O,P; 36%, n=146). Expression of rostral neural plate markers, emx3 (Fig. 1Q,R; 56%, n=32) and six3b (Fig. 1S,T; 62%, n=77), are significantly reduced or completely lost at tail bud stage in injected embryos. Expression of wnt8b is expanded rostrally into what remains of the forebrain by mid-somitogenesis (Fig. 1U,V; 39%, n=33). We obtain similar although generally less severe phenotypes with antisense morpholinos against lhx5. In the lhx5 morpholino-injected embryos, the six3b expression domain is slightly reduced at tail bud stage (48%, n=67), pax6a in the posterior optic vesicle is significantly reduced at the 12-somite stage (Fig. 1W,X; 70%, n=84) and rostrolateral pax2a expression expands into the posterior-medial optic vesicle (Fig. 1Y,Z; 50%, n=96). The lhx5 morpholino-injected embryos later develop small heads with small eyes (73%, n=175). Injection of a second morpholino that blocks the splicing of lhx5 transcripts had similar effects on forebrain development (see Fig. S1A-D in the supplementary material; pax6a, 72%, n=32; pax2a, 78%, n=32).
The weaker effect of the morpholinos, when compared with the dominant
interfering construct, may be due to an incomplete block of Lhx5 function.
From RT-PCR analysis, we estimate that the lhx5 splice-blocking
morpholino reduces lhx5 mRNA level to about 8% of control levels
(Fig. S1H). Thus, it is possible that residual lhx5 mRNA may have
given rise to sufficient Lhx5 protein to allow a partial development of the
forebrain in morpholino-injected embryos. Similarly, we cannot exclude the
possibility that Lhx5-En may interfere with other LIM homeodomain factors by
heterodimer formation between LIM domains
(Hobert and Westphal, 2000).
Nevertheless, these results together support the conclusion that Lhx5 is
required for forebrain development.
Lhx5 activity rescues forebrain deficiencies caused by ectopic Wnt signaling
The expansion of forebrain we see in lhx5 mRNA-injected embryos is
also observed in embryos lacking wnt8a gene function
(Erter et al., 2001;
Lekven et al., 2001), and in
embryos injected with Wnt inhibitors such as dkk1 mRNA
(Hashimoto et al., 2000). In
addition, the expression domains of emx3 and six3b are
expanded in embryos injected with wnt8b morpholinos
(Houart et al., 2002;
Kim et al., 2002). Conversely,
the compromised forebrain development caused by the inhibition of Lhx5
function is similar to defects in embryos with increased Wnt signaling caused
by wnt8a mRNA injection (Kelly et
al., 1995), or by mutations in the masterblind
(axin1) gene (Heisenberg et al.,
2001; van de Water et al.,
2001). We thus examined interactions between Lhx5 activity and Wnt
signaling (Fig. 2).
|
). By
contrast, when we co-inject lhx5 mRNA with wnt8a mRNA, the
effect of Wnt8a is suppressed; the majority of the injected embryos (57%,
n=107) develop two eyes. As a control, we generated a truncated
lhx5 construct in which the transcriptional activation domain of Lhx5
is missing. When the truncated lhx5 mRNA is co-injected with
wnt8a mRNA, very few (14%, n=64) of the injected embryos
form two eyes.
We also examined the modulation of Wnt signaling by Lhx5 in mbl
(axin1) mutant embryos. Previous studies suggested that the
degradation of ß-catenin is less efficient and Wnt signaling is
overactivated in mbl-/- (axin1) embryos when
compared with controls (Heisenberg et al.,
2001; van de Water et al.,
2001).
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Secreted Frizzled-related proteins, Sfrp1a and Sfrp5, antagonize Wnt signaling in the forebrain
To determine the mechanism by which Lhx5 inhibits Wnt signaling, we
identified secreted frizzled-related proteins (Sfrps)
(Jones and Jomary, 2002;
Kawano and Kypta, 2003) as
downstream targets of Lhx5. Sfrps are important Wnt regulators. Sfrps can bind
directly to Wnts (Dennis et al.,
1999; Lin et al.,
1997; Uren et al.,
2000; Xu et al.,
1998) and they are dynamically expressed during development
(Pera and De Robertis, 2000;
Terry et al., 2000).
Based on the available genome sequence, we cloned five zebrafish Sfrp
genes. All five Sfrps fall into a phylogenetic subgroup that includes Sfrp1,
Sfrp2 and Sfrp5 (Fig. 3). On
the basis of our mapping results (data not shown), we suggest that
sfrp1a and sfrp1b have arisen from the extra genome
duplication that occurred in the ray fin fish lineage
(Postlethwait et al., 1998).
Currently it is unknown how many Sfrp orthologs are present in the zebrafish
genome.
We concentrated our studies on sfrp1a and sfrp5 because
their transcripts are present in the nervous system, as shown by whole-mount
in situ hybridization. Transcripts for sfrp2 and sfrp2l are
found in cells that give rise to muscle, whereas sfrp1b is expressed
in a region surrounding the yolk extension (data not shown)
(Tendeng and Houart,
2006).
Previously, it was shown that overexpression of Sfrp1 blocks the dorsal
axis duplication induced by xwnt8 mRNA injection in Xenopus
embryos, suggesting that Sfrp1 antagonizes Wnt signaling
(Finch et al., 1997). To test
whether Sfrp1a or Sfrp5 similarly antagonizes Wnt signaling in zebrafish, we
injected sfrp1a or sfrp5 mRNAs together with wnt8a
mRNA. Both Sfrp1a and Sfrp5 rescue the Wnt8a-induced eyeless phenotype very
effectively (Fig. 4A). In
sfrp1a and wnt8a co-injected embryos, 80% of the embryos
develop two eyes (n=64); co-injection of sfrp5 with
wnt8a rescues eye development in 93% of the injected embryos
(n=70). Both Sfrps also rescue the early dorsalization of
wnt8a-injected embryos equally well, suggesting that they have
similar effects on Wnt signaling during early development
(Fig. 4A).
Similar to Lhx5, overexpression of Sfrp1a or Sfrp5 promotes forebrain development. We injected sfrp1a or sfrp5 mRNA into one- to two-cell stage embryos. We find that by mid-somitogenesis stages, sfrp5-injected embryos exhibit enlarged forebrains, whereas forebrain enlargement is less pronounced in sfrp1a-injected embryos (see Fig. S2 in the supplementary material). At the end of gastrulation, sfrp1a overexpression causes an expansion of the emx3 and six3b expression domains in a small percentage of injected embryos (Fig. 4C,F; six3b, 10%, n=42; emx3, 22%, n=41), whereas sfrp5 overexpression results in more robust expansion of the emx3 and six3b domains (Fig. 4D,G; six3b, 60%, n=40; emx3, 53%, n=40, respectively).
We also examined whether the overexpression of sfrp1a or
sfrp5 can rescue forebrain development in mbl-/-
embryos. sfrp1a, sfrp5 or sfrp1a plus sfrp5 mRNA
injection fails to restore eye development to mbl-/-
embryos when scored at 3 days of development. Nevertheless, emx3
expression is fairly well rescued in mbl-/- embryos
injected with sfpr1a and sfrp5 mRNA together
(Fig. 4J; 58%,
nmbl=12) or with sfrp5 mRNA alone (62%,
nmbl=13). Expression of six3b is also partially
rescued in mbl-/- embryos injected with sfrp1a
and sfrp5 together (Fig.
4M; 60%, nmbl=10) or with sfrp5 mRNA
alone (67%, nmbl=12). sfrp1a mRNA injections fail
to rescue emx3 or six3b expression in
mbl-/- embryos. It is unclear what factors are responsible
for the differences between sfrp1a and sfrp5 in these
assays. There are few functional studies of Sfrp5. Sfrp1 function is complex,
involving all three branches of the Wnt signaling pathway
(Dennis et al., 1999;
Esteve and Bovolenta, 2006;
Esteve et al., 2004;
Lin et al., 1997;
Rodriguez et al., 2005;
Satoh et al., 2006;
Xu et al., 1998). Differences
in RNA stability may also contribute to differences between the overexpression
effects of sfrp1a and sfrp5.
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Lhx5 function is required for the expression of sfrp1a in the forebrain. In lhx5 morpholino-injected embryos, sfrp1a expression in the presumptive forebrain is significantly reduced or completely lost, whereas expression in hindbrain and posterior mesoderm is relatively unaffected (Fig. 5E,F; 71%, n=93 and 51%, n=67, respectively). Later in the pharyngula period (prim-7 stage), sfrp1a is strongly expressed in the forebrain (Fig. 5G) and injection of the lhx5 morpholino significantly reduces this expression in a dose-dependent manner (Fig. 5H,I; 2 ng morpholino, 80%, n=96; 4 ng morpholino, 77%, n=66). The lhx5 splice-blocking morpholino similarly reduces sfrp1a expression at this stage (data not shown).
Lhx5 regulates Sfrp1a expression cell autonomously. To demonstrate this, we transplanted animal pole cells from labeled midblastula stage donor embryos to late blastula stage host embryos. The transplanted donor cells (red labeled) express sfrp1a (blue labeled and arrowheads) in the late gastrula, when they are distributed in the presumptive forebrain of host embryos (Fig. 5J; 100%, n=20). We then injected the donor embryos with the dominant interfering lhx5-en mRNA or the lhx5 translation-blocking morpholino before transplanting the donor cells into the uninjected host embryos. The transplanted lhx5-en-expressing donor cells do not express sfrp1a even when they are located in the presumptive forebrain of host embryos (Fig. 5K; 65%, n=20), whereas morpholino-injected donor cells have a reduced sfrp1a expression (Fig. 5L, 33%, n=12). Similarly, we transplanted donor cells from lhx5 mRNA-injected embryos into lhx5 morpholino-injected host embryos. The transplanted lhx5-expressing donor cells also express sfrp1a in the presumptive forebrain, whereas neighboring lhx5 morpholino-containing cells do not regain sfrp1a expression (Fig. 5M; 87%, n=30). Later during segmentation stages (18-somite stage), transplanted lhx5 mRNA-injected donor cells continue to express sfrp1a in the forebrain of lhx5 morpholino-injected host embryos (data not shown).
Lhx5 binds to the sfrp1a promoter. We identified sfrp1a
promoter elements that are sufficient for sfrp1a expression in
forebrain (Fig. 5N). We
injected a series of deletion constructs of the sfrp1a upstream
sequence fused to GFP-coding sequence into zebrafish embryos and assayed the
transient GFP expression in forebrain regions (see Fig. S3 in the
supplementary material). We find that a 2.7 kb sfrp1a upstream
sequence is necessary and sufficient to drive high levels of GFP expression in
forebrain. We then broke up the sfrp1a promoter into seven
overlapping fragments and co-injected each fragment together with the
sfrp1a (-371) basal promoter fused to the GFP-coding sequence
(Muller et al., 2000). A 680
bp fragment about 1500 bp upstream of the Sfrp1a-coding sequence is most
likely to be responsible for sfrp1a expression in forebrain
(Fig. 5N). To test whether Lhx5
binds to this fragment, we used formaldehyde-based in vivo cross-linking and
chromatin immunoprecipitation (Fig.
5O). We find that the sfrp1a promoter element is
significantly enriched in Lhx5-associated chromatin, whereas upstream
sequences of the housekeeping genes aldolase a and bactin2
(human actin beta gene, ACTB ortholog) are not significantly enriched in
Lhx5-associated chromatin. These results indicate that Lhx5 binds to the
sfrp1a promoter element that directs Sfrp1a expression in the
forebrain.
Lhx5 regulates sfrp5 expression. sfrp5 expression starts by the end of gastrulation in the presumptive forebrain and hindbrain (data not shown). By the 5-somite stage, sfrp5 expression is strong in the presumptive forebrain (Fig. 5P). In embryos injected with lhx5 mRNA, we note little change in sfrp5 transcript levels at tail bud stage (data not shown), but by the 5-somite stage, sfrp5 expression is expanded in the forebrain (Fig. 5Q; 52%, n=61). The expansion of sfrp5 expression correlates with the increase in size of the forebrain from this stage onwards. In lhx5 morpholino-injected embryos, sfrp5 expression is reduced at the 5-somite stage (Fig. 5R; 70%, n=82). Thus, Lhx5 is apparently required for normal sfrp5 expression, although it is not sufficient to activate sfrp5 expression. Later in development, sfrp5 transcripts are distributed in the forebrain in a pattern similar to, but distinct from, the expression pattern of sfrp1a. Expression of sfrp5 is still reduced in lhx5 morpholino-injected embryos at these later stages (Fig. 5T,U; 65%, n=62 and 57%, n=67, respectively).
|
Lhx5 regulates Wnt signaling
We used expression of the endogenous axin2 gene as a Wnt pathway
reporter gene. The mouse Axin2 gene contains multiple TCF-binding
sites in its promoter and introns, and is a direct target of the canonical Wnt
signaling pathway (Jho et al.,
2002). We identified multiple TCF-binding sites in the zebrafish
axin2 genomic sequence (data not shown) and examined the expression
of axin2 in response to changes in Wnt signaling. We find that the
zygotic axin2 gene expression pattern closely matches Wnt signaling
activity. At blastula stages, axin2 is expressed in the marginal zone
(Fig. 6B), where wnt8a
and other Wnt genes are known to be expressed
(Kelly et al., 1995). When Wnt
signaling is ectopically activated by wnt8a mRNA injection, the
entire blastula expresses axin2
(Fig. 6C; 93%, n=96),
and this ubiquitous activation of axin2 expression persists during
gastrula stages (data not shown)
(Weidinger et al., 2005). By
the end of the gastrulation, axin2 is normally restricted to
posterior tissues; presumptive forebrain exhibits little axin2
expression (Fig. 6D). In
mbl-/- embryos that have expanded Wnt signaling
(Heisenberg et al., 2001;
Houart et al., 2002),
axin2 expression expands rostrally into the presumptive forebrain
(Fig. 6E; 21%, n=63).
These results demonstrate that the zebrafish axin2 gene can be used
as a Wnt pathway reporter.
Lhx5 regulates axin2 expression. In lhx5 morpholino-injected embryos, axin2 expression is not significantly affected during gastrulation (data not shown). However, we observe elevated axin2 expression in the forebrain at the 16-somite stage in lhx5 morpholino-injected embryos (Fig. 6G,I; 53%, n=68), consistent with the view that Lhx5 negatively regulates Wnt signaling. When the dominant interfering lhx5-en construct is injected, axin2 expression is ectopically activated in rostral ectoderm during gastrulation (Fig. 6K; 80%, n=79), indicating ectopic activation of Wnt signaling. By contrast, when sfrp1a or sfrp5 mRNA is co-injected together with lhx5-en mRNA, the ectopic expression of axin2 is blocked such that rostral ectoderm is largely free of axin2 expression (Fig. 6L,M; 59%, n=64 and 67%, n=63, respectively).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wnt proteins play crucial roles during nervous system development
(Ciani and Salinas, 2005;
Wilson and Edlund, 2001;
Wilson and Houart, 2004).
During gastrula stages, Wnts expressed by the posterior paraxial mesoderm
(Kelly et al., 1995) are
thought to act as graded signals that caudalize the neural plate
(McGrew et al., 1995;
Nordstrom et al., 2002). In
zebrafish, mutations in the headless (tcf7l1a) or
masterblind (axin1) genes, which both encode negative
regulators of Wnt signaling, truncate the prosencephalon
(Heisenberg et al., 2001;
Kim et al., 2000;
van de Water et al., 2001). We
used axin2 expression as a marker of Wnt signaling; rostral ectoderm
normally exhibits little axin2 expression, and overactivation of Wnt
signaling by injection of wnt8a mRNA
(Fig. 6C) or
masterblind (axin1) mutation results in the ectopic
activation of axin2 (Fig.
6E). Dominant interference with Lhx5 function also results in the
ectopic activation of axin2 in the rostral ectoderm
(Fig. 6K), whereas Sfrp mRNA
injection reduces ectopic axin2 expression and the forebrain defects
that result from loss of Lhx5 function
(Fig. 6L,M). Therefore, it is
likely that Lhx5-regulated Sfrp expression is an important part of the
mechanism that normally suppresses Wnt signaling in the rostral ectoderm
during early development.
|
; Tolwinski and Wieschaus,
2004). Therefore, it is somewhat surprising that changes in
extracellular Wnt signaling affect mbl-/- embryos.
Consistent with our results, however, the reduction of wnt8b function
by morpholino knockdown or the transplantation of tlc-expressing
cells restores emx3 expression in mbl-/- embryos,
suggesting that extracellular Wnts contribute to the loss of forebrain in
mbl-/- embryos (Houart
et al., 2002). It is unclear whether the response to extracellular
wnt8b levels in mbl-/- embryos is due to
maternally deposited Axin1 (Heisenberg et
al., 2001) or to a residual function of the
mbltm213 allele. Axin1 is present only at very low levels
in Xenopus egg extracts (Lee et
al., 2003) and the degradation of Axin1 is regulated by Wnt
signaling (Tolwinski and Wieschaus,
2004). These features, if conserved in the zebrafish Wnt signaling
pathway, raise the possibility that the missense mutation of
mbltm213 is a hypomorphic allele.
|
). The
presumptive eye field is specified as part of the forebrain anlage and,
subsequently, eye vesicles evaginate from the forebrain to form eyes. Wnt
signaling is implicated in eye formation
(Esteve and Bovolenta, 2006;
Wilson and Houart, 2004) and
there is, apparently, a cell-autonomous requirement for Axin1 function for eye
development (Heisenberg et al.,
1996; Houart et al.,
2002). Our studies show that the overexpression of Sfrps does not
restore eye development in mbl-/- embryos and results in
limited rescue of six3b expression. By contrast, overexpression of
Lhx5 largely restores six3b expression and partially rescues eye
development in mbl-/- embryos. Although we cannot exclude
the possibility that factors such as mRNA or protein stability may contribute
to the different outcomes, a parsimonious model might include other Lhx5
downstream factors that contribute to the restoration of eye development in
mbl-/- embryos (Fig.
7).
|
).
Overexpression of Six3 in zebrafish or medaka leads to the enlargement of
forebrain structures (Kobayashi et al.,
2001; Loosli et al.,
1999), and inhibition of Six3 function in mouse or medaka results
in truncation of the prosencephalon (Carl
et al., 2002; Lagutin et al.,
2003). Remarkably, overexpression of mouse Six3 in zebrafish
rescues the forebrain deficiency of headless (tcf7l1a)
mutant embryos (Lagutin et al.,
2003). Thus, Six3 and Lhx5 appear to have similar functions,
although there are differences. In zebrafish, six3 expression is
initiated in the rostral ectoderm at late gastrulation stages, whereas
lhx5 is already expressed by the onset of gastrulation. We find that
six3 expression in masterblind (axin1) mutant
embryos is significantly reduced at the end of gastrulation and that
overexpression of Lhx5 can largely restore Six3 expression and rescue the
forebrain deficiency. Thus, although both Lhx5 and Six3 suppress Wnt signaling
and promote forebrain development, their molecular targets and the timing of
their activities probably differ. A recent study showed that Six3 also
functions to control cell proliferation by sequestering Geminin from Cdt1, a
key component in the assembly of the pre-replication complex
(Del Bene et al., 2004). Lhx2
may function downstream of Six3 to regulate cell proliferation in the
developing forebrain (Ando et al.,
2005). Currently it is unknown whether Lhx5 plays a similar role
in cell proliferation.
Function of Lhx5 in vertebrate development
Lhx5 belongs to the Lin-11 group of LIM-homeodomain factors
(Hobert and Westphal, 2000).
Vertebrate Lhx5 orthologs share conserved expression patterns and are
implicated in the establishment of the prosomeric organization of the
forebrain (Bachy et al., 2001).
In frogs or fish, lhx5 is broadly expressed by early gastrulation
stages. Inhibition of Lhx5 function in frogs causes the dissociation of
rostral ectodermal cells during gastrulation
(Houston and Wylie, 2003), and
our studies in zebrafish suggest that Lhx5 regulates Wnt antagonism in the
gastrula. In mice, however, no gastrulation defects have been reported in
Lhx5 knockout animals (Zhao et
al., 1999). Published data show that the earliest mouse
Lhx5 expression is detected in the rostral neural plate at E8.0 when
gastrulation is ending and the late head fold is forming
(Sheng et al., 1997). It is
unclear whether the absence of gastrulation defects in Lhx5 knockout
mice is due to a late onset of Lhx5 expression in rostral ectoderm or
to unknown mechanisms that compensate for loss of Lhx5 function.
Mouse Sfrp2 is broadly expressed in the ectoderm
(Mukhopadhyay et al., 2003)
and Sfrp5 is expressed in the rostral visceral endoderm during
gastrulation (Finley et al.,
2003). Thus, either Sfrp2 or Sfrp5 may antagonize Wnt signaling
and allow forebrain development in the absence of Sfrp1.
Lhx5 shares high sequence identity with Lhx1
(Hobert and Westphal, 2000).
Vertebrate Lhx1 orthologs share conserved expression in the
mesoendoderm at early gastrulation stages
(Barnes et al., 1994;
Taira et al., 1992;
Toyama and Dawid, 1997). In
mouse and frog embryos, Lhx1 activity is required in the mesoendoderm for the
formation of anterior head structures
(Hukriede et al., 2003;
Shawlot and Behringer, 1995;
Tam et al., 2004). If
zebrafish Lhx1 plays a similar role in anterior head development, the severe
phenotype caused by Lhx5-En may be due partly to its interference with Lhx1
function. Interestingly, Sfrp1 expression is reduced in the anterior
mesoendoderm in mouse Lhx1 knockout embryos (see
Satoh et al., 2006). In our
studies, however, mesoendoderm sfrp1a expression is retained in
lhx5-en mRNA-injected embryos
(Fig. 5C). Thus, we can
conclude that Lhx5-En does not significantly interfere with the Lhx1
regulation of sfrp1a expression. Nevertheless, we cannot exclude the
possibility that Lhx5-En may interfere with other functions of Lhx1 in the
mesoendoderm. Further studies are needed to determine the functional
relationship between Lhx5 and Lhx1 in zebrafish forebrain development.
Lhx5 regulation of Sfrps has not been previously reported. In mouse,
Lhx5 knockout results in excess neural-precursor cell proliferation
and migration defects during hippocampus formation
(Zhao et al., 1999). Because
Wnt signaling induces mitogenic activity in the nervous system
(Megason and McMahon, 2002)
and Wnt3a positively regulates mouse hippocampal development
(Lee et al., 2000), it is
possible that Wnt signaling is elevated in the hippocampus of Lhx5
knockout mice. Sfrp1 is also expressed in the mouse hippocampus.
Thus, Lhx5 regulation of Wnt antagonists may be a conserved mechanism that
also functions during development of the hippocampus.
Previous studies have shown that members of the LIM homeodomain protein
family play crucial roles in the terminal differentiation of neuronal cells
(Hobert and Westphal, 2000;
Jessell, 2000;
Shirasaki and Pfaff, 2002). In
addition to its early expression in the presumptive forebrain, lhx5
is also expressed later in subsets of interneurons
(Toyama et al., 1995). A
recent study showed that mouse Lhx5 marks neurons in the rostral part of the
medial amygdala that project primarily to the hypothalamic nuclei associated
with defensive behavior (Choi et al.,
2005). Further studies are needed to determine whether Lhx5 acts
in the context of Sfrps and Wnt signaling to regulate the terminal
differentiation of these interneuron cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/16/3191/DC1
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ACKNOWLEDGMENTS |
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