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. 2003 Feb 1;17(3):368-79.
doi: 10.1101/gad.1059403.

Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development

Oleg V Lagutin  1 , Changqi C Zhu, Daisuke Kobayashi, Jacek Topczewski, Kenji Shimamura, Luis Puelles, Helen R C Russell, Peter J McKinnon, Lilianna Solnica-Krezel, Guillermo Oliver
Affiliations

Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development

Oleg V Lagutin et al. Genes Dev. .

Abstract

In vertebrate embryos, formation of anterior neural structures requires suppression of Wnt signals emanating from the paraxial mesoderm and midbrain territory. In Six3(-/-) mice, the prosencephalon was severely truncated, and the expression of Wnt1 was rostrally expanded, a finding that indicates that the mutant head was posteriorized. Ectopic expression of Six3 in chick and fish embryos, together with the use of in vivo and in vitro DNA-binding assays, allowed us to determine that Six3 is a direct negative regulator of Wnt1 expression. These results, together with those of phenotypic rescue of headless/tcf3 zebrafish mutants by mouse Six3, demonstrate that regionalization of the vertebrate forebrain involves repression of Wnt1 expression by Six3 within the anterior neuroectoderm. Furthermore, these results support the hypothesis that a Wnt signal gradient specifies posterior fates in the anterior neural plate.

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Figures

Figure 1
Figure 1
Targeted disruption of Six3. (A) Six3 was inactivated by the in-frame insertion of a β-galactosidase–neomycin resistance cassette in the NcoI–XhoI site located 22 amino acids downstream of the first initiation methionine. (B) In embryonic stem cells, the targeted Six3 locus was identified by Southern blot analysis of genomic DNA digested with HindIII and hybridized to a 0.5-kb external genomic fragment. The 13.0-kb fragment originated from the wild-type allele, and the ∼3.7-kb fragment originated from the targeted allele.
Figure 1
Figure 1
Targeted disruption of Six3. (A) Six3 was inactivated by the in-frame insertion of a β-galactosidase–neomycin resistance cassette in the NcoI–XhoI site located 22 amino acids downstream of the first initiation methionine. (B) In embryonic stem cells, the targeted Six3 locus was identified by Southern blot analysis of genomic DNA digested with HindIII and hybridized to a 0.5-kb external genomic fragment. The 13.0-kb fragment originated from the wild-type allele, and the ∼3.7-kb fragment originated from the targeted allele.
Figure 2
Figure 2
Inactivation of Six3 results in the absence of the eyes and nose and leads to postnatal lethality. Wild-type (A,C), Six3+/− (E), and Six3−/− (B,D,F) embryos were used. Staining of bone (red) and cartilage (blue) of wild-type (C) and Six3-null (D) embryos revealed substantial stunting of the rostral skull (arrow) and maxillofacial derivatives; the mandible is also indicated (arrowhead). (E) In an E12.5 Six3-heterozygous embryo, X-gal staining recapitulated the normal pattern of Six3 expression in the eyes, nose (black arrowhead), midbrain, pretectal tegmentum (the white arrow is placed at the boundary between midbrain and pretectum, just caudal to the posterior commissure), ZLI (white arrowhead), and rostral ventral thalamus (red arrow). (F) In the Six3−/− littermates, the residual X-gal staining was detected throughout the midbrain and forebrain tegmentum, the pretectum (weakly stained), and in transverse bands, which may correlate with the ZLI (white arrowhead) and rostral part of the ventral thalamus (red arrow).
Figure 3
Figure 3
Six3−/− embryos lack a rostral forebrain. Whole-mount in situ hybridization of E9.5–E10.0 wild-type and Six3-null embryos. Bf1 (A) and Emx2 (C) were expressed in the telencephalic vesicles of the wild-type embryos but not in the Six3−/− littermates (B,D). Fgf8 was expressed in the commissural plate (arrow) and isthmus (arrowheads) of wild-type embryos (E) but only in the isthmus (arrowhead) of Six3−/− embryos (F). In wild-type embryos (G), Pax6 expression was localized in the telencephalon and alar diencephalon, including the optic vesicles; in the Six3−/− embryos (H), Pax6 expression was normal, except in the missing telencephalic vesicles and eyes. (I) In wild-type embryos, Nkx2.1 was expressed in the posterior hypothalamus (arrowhead), infundibular hypothalamus (white arrow), and basal telencephalon (black arrow). (J) In the Six3-null littermates, only the most caudal portion of the wild-type Nkx2.1 ventral expression domain was present in the posterior hypothalamus. (K) At E10.0, Shh expression extended along the ventral diencephalon into the postchiasmatic forebrain (black arrow) and the ZLI (red arrowhead) of wild-type embryos. (L) The rostral area of Shh expression in the basal plate of the hypothalamus was absent in Six3−/− embryos; however, that of the ZLI persisted (red arrowhead). (M) In E10.0 wild-type embryos, Wnt1 was expressed in the roof of the caudal diencephalon and mesencephalon and in a transverse band in the isthmus (arrow). (N) In Six3-null embryos, the Wnt1 expression in the diencephalic roof was rostrally expanded, but that in the isthmus appeared normal. Compared with the expression of the midbrain marker Pax3 in E10.0 wild-type embryos (O), that in the truncated Six3-null forebrain was expanded rostrally in the dorsal alar plate (P). (Q) En2 was expressed across the midbrain-hindbrain boundary in E10.0 wild-type embryos. (R) Although apparently expanded, the pattern of En2 expression was maintained in E10.0 Six3-null littermates. (S) In wild-type E10.0 embryos, Otx2 was expressed in the forebrain and midbrain. (T) In Six3-null littermates, Otx2 expression extended to the anterior end of the mutant forebrain.
Figure 4
Figure 4
The patterning of the anterior visceral endoderm (AVE) and anterior neuroectoderm during early development is normal in Six3-null embryos; lack of rostral forebrain is detected at the early (4–6) somite stage. (A) Whole-mount in situ hybridization of ∼E7.0–E7.5 wild-type embryos showed strong expression of Hesx1 in the anterior endoderm (black arrow) and weak expression in the overlying epiblast (anterior ectoderm) from which the future forebrain will develop (red arrowhead). (B) A similar pattern of Hesx1 expression was observed in Six3-mutant littermates. (C) At the neural-plate stage (E7.5–E8.0), Hesx1 expression was more abundant in the anterior neuroectoderm (future forebrain region) of the wild-type epiblast (red arrowhead). (D) No obvious alterations were detected in the mutant littermates. (E) At the 1–2-somite stage, Hesx1 expression was observed in the most anterior neuroectoderm of wild-type embryos (black arrowhead) including the floor of the diencephalic portion of the primitive forebrain. (F) A reduction in the level of Hesx1 expression was initially detected in the margins of the anterior neuroectoderm of Six3-null littermates. (G) Scanning electron micrographs of E8.5 embryos at the 5–8-somite stage showed that the optic pit evaginations (white arrowhead) and the anterior neural ridge (white arrow) evident in the wild-type embryos were absent in Six3-null littermates (H), as a result of the lack of rostral forebrain; the midbrain region appeared to be anteriorly expanded. At the 4–6-somite stage (I), Hesx1 was widely expressed in the rostral region of the anterior neuroectoderm (future forebrain) of wild-type embryos; in Six3-null littermates (J), Hesx1 expression was restricted to a narrow medial domain in the most anterior part of the mutant neural plate. (K) Rx was expressed in the anterior neuroectoderm of wild-type embryos but was barely detectable in the Six3-null littermates (L). Fgf8 was expressed in the anterior neural ridge (ANR; arrow) and isthmus (arrowhead) of wild-type embryos (M); Fgf8 was not detected in the rostral neuroectoderm (ANR is missing) but appeared unaffected in the isthmus (arrowhead) of the Six3-null embryos (N). (O) Bf1 was expressed in the anterior neural plate and underlying non-neural ectoderm of wild-type embryos. (P) Bf1 expression remained only in the nonneural ectoderm of Six3-null littermates. (Q) During the early (3–5) somite stage, Pax3 expression demarcated the prospective alar midbrain region in wild-type embryos. In Six3-null littermates (R), Pax3 expression had already extended beyond its normal midbrain boundary into the anterior part of the neuroectoderm, suggesting that in the mutant embryos this region has adopted a midbrain identity; Pax3 remains negative in the most paramedial region (red arrowhead), an area in which Hesx1 expression (J) was still detected.
Figure 5
Figure 5
Six3 represses Wnt1 gene activity during vertebrate head development. (A) At the early (1–2) somite stage, Wnt1 expression was almost undetectable in wild-type embryos. (B) Weak expression of Wnt1 was detected in the developing midbrain of the Six3-heterozygous littermates. (C) The level of Wnt1 expression was the highest in the comparable region of the Six3-null littermates. (D) A few hours later (6–8-somite stage), normal Wnt1 expression demarcated the developing midbrain (arrow). (E) In the Six3-null littermates, Wnt1 expression in the midbrain territory (arrow) was maintained, and the ectopic rostral expansion of its expression was quite evident at this stage anterior to the midbrain territory (arrowheads). (F) Green fluorescent protein was expressed throughout the electroporated right side of the CNS of HH stage 8–9 chicken embryos. (G) Whole-mount in situ hybridization of Six3-electroporated embryos showed normal Wnt1 expression along the CNS on the nonelectroporated left side; no Wnt1 expression was observed on the Six3-electroporated contralateral side (arrowheads). (G‘) A magnification of the electroporated embryo shown in G. (H) EMSA assay shows that bacteria-expressed GST–Six3 fusion protein binds to the Wnt1 promoter elements I and II (lanes 3,8) strongly, very weakly to element III (lane 13), and not at all to element IV (data not shown). No specific binding was detected when using as negative controls either probes I, II, and III alone (lanes 1,6,11), or probes I, II, and III together with GST protein (lanes 2,7,12). The binding of Six3 to probes I and II can be competed when using 400× molar excess of their corresponding wild-type unlabeled oligonucleotides (lanes 4,9), but not when using similar amounts of unlabeled mutated oligonucleotides (lanes 5,10). (Lane 18) The specific binding of the GST–Six3 fusion protein to the 40-bp Wnt1 3′-enhancer element. This element alone (lane 16) or together with GST protein (lane 17) does not show any specific binding. GST–Six3 fusion protein bound to this Wnt1 enhancer element can be competed when using 100× molar excess of unlabeled wild-type oligonucleotide (lane 19), but not when using a mutated form (lane 20). The binding complex is supershifted by either an anti-Six3 antibody or anti-GST antibody (lanes 21,22). *, the binding complex of GST–Six3 and DNA; arrowhead, supershifted binding complex using anti-Six3 antibody; arrow, supershifted binding complex using anti-GST antibody. (I) An ∼700-bp DNA fragment located 5′ of the Wnt1 transcriptional initiation site includes seven clustered regions containing putative Six3 DNA-binding motifs (*). Primers A and B were used for the ChiP assay. (J) ChiP assay on E8.5 dissected head and trunk regions of wild-type embryos showing in vivo recruitment of Six3 to the Wnt1 5′-promoter (primers A/B) and 3′-enhancer (primers C/D) regions. No recruitment of Six3 was detected when using primers (primers E/F) against an unrelated 5′ genomic region of Wnt1. Specific PCR amplification was only observed when using DNA extracted from the head region.
Figure 6
Figure 6
Ectopic murine Six3 represses wnt1 expression and rescues forebrain deficiency in hdl mutant zebrafish embryos. (A) Whole-mount in situ hybridization analysis (Marlow et al. 1998) of wnt1 expression in wild-type embryos 10 h postfertilization. (B) Injection of synthetic murine Six3 mRNA into wild-type embryos at the one-cell stage (Thisse and Thisse 1998) repressed endogenous wnt1 expression in 90% of the embryos (n = 164). (C) wnt1 (arrowhead) and six3 (arrow) expression was observed in control (uninjected) hdl embryos. In these embryos, an enlarged wnt1 expression domain was observed in 91% of cases (n = 45). (D) Mouse Six3 was overexpressed in hdl embryos as described (Thisse and Thisse 1998; van de Water et al. 2001); 2% of the injected embryos maintained an enlarged Wnt1 expression domain, whereas the remaining 98% exhibited normal, reduced, or absent expression (n = 63). (E) The lack of eyes normally seen in control hdl mutants 2 d postfertilization was suppressed in a mutant sibling injected with mouse Six3 mRNA (F). Of the control hdl embryos (n = 153), 38% lacked eyes and 62% exhibited small eyes. In contrast, 90% of the mutant embryos that received mouse Six3 mRNA injections (n = 89) had normal eyes, 5% had small eyes, and 5% had abnormal morphology.
Figure 7
Figure 7
(A) A Gsk3 expression vector was electroporated on one side of the midbrain (m) and hindbrain (h) regions of HH stage 5 chicken embryos. In situ hybridization of HH stage 8–9 embryos revealed ectopic Six3 activation throughout the electroporated side of the midbrain and hindbrain regions (arrowheads); normal Six3 expression was seen in the forebrain (f) region. (B) Wnt3A electroporation repressed Six3 expression in one side of the anterior neuroectoderm.
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References

    1. Acampora D, Avantaggiato V, Tuorto F, Simeone A. Genetic control of brain morphogenesis through Otx gene dosage requirement. Development. 1997;124:3639–3650. - PubMed
    1. Bachiller D, Klingensmith J, Kemp C, Belo JA, Anderson RM, May SR, McMahon JA, McMahon AP, Harland RM, Rossant J, et al. The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature. 2000;403:658–661. - PubMed
    1. Belo JA, Bouwmeester T, Leyns L, Kertesz N, Gallo M, Follettie M, De Robertis EM. Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech Dev. 1997;68:45–57. - PubMed
    1. Camus A, Davidson BP, Billiards S, Khoo P, Rivera-Perez JA, Wakamiya M, Behringer RR, Tam PP. The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embryo. Development. 2000;127:1799–1813. - PubMed
    1. Crossley PH, Martin GR. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development. 1995;121:439–451. - PubMed

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