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Rhinn M
,
Lun K
,
Ahrendt R
,
Geffarth M
,
Brand M
.
Abstract
BACKGROUND: Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, on the other hand, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, we used a combination of gain- and loss-of-function experiments.
RESULTS: We found that ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. We further found that ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. Our work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, we show that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. We propose that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain.
CONCLUSION: Our results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, we have shown that gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning.
Figure 1. Overexpression of gbx1 induces posterior cell fate. (A) Wild-type (WT) embryo at 24 h and (B) after injection of 200 pg gbx1 mRNA; anterior brain structures are severely reduced. The ear is indicated by an arrowhead. (C, D) Staining of the forming axon tracts with an anti-acetylated tubulin antibody at 24 h; dorsal views, with anterior to the top. In the gbx1-injected embryo the hindbrain is severely enlarged compared to WT (arrowheads). (E, F) Duplications of ear structures are frequently observed (arrows). (G-I') Series of embryosinjected with different doses of gbx1 mRNA (200 and 500 pg), analyzed at the tailbud stage after in situ hybridization with otx2 (blue) and krox20 (red). The otx2 domain progressively disappears and the krox20 domains shift to more anterior regions. (J) Dose-dependent gbx1 overexpression phenotypes. Higher concentrations (>500 pg) did not increase the observed phenotype. (A-D, K-M) Lateral views; (E-H, K'-M') dorsal views.
Figure 2. gbx1 overexpression affects midbrain, midbrain-hindbrain boundary (MHB) and hindbrain. (A, A') Control embryo stained for six3 (blue) and otx2 (red);. (B, B') gbx1-injected embryo. The near absence of otx2-positive cells posteriorly to the six3-expressing domain indicates a loss of the midbrain territory in the gbx1-injected embryos. (C, C') Control embryo stained for emx1 (blue) and otx2 (red). (D, D') gbx1-injected embryo. The emx1 domain corresponding to forebrain remains robust, whereas the otx2 domain corresponding to midbrain is lost. (E) Wild-type (WT) embryo after in situ hybridization (ISH) with pax2.1 and (E') with pax2.1 and otx2 (red). (F, F') gbx1-injected embryos; pax2.1 is expressed ectopically in the anterior of the embryo. The MHB is shifted anteriorly as indicated by green arrowheads. Also, the distance between the posterior pax2.1 domain and the anterior domain is increased (black bars in (E, F)). (G) WT embryo after ISH with fgf8 and (G') with fgf8 and otx2 (red). (H, H') gbx1-injected embryos; fgf8 is expressed ectopically in the anterior of the embryo. The MHB is shifted anteriorly as the distance between this expression domain and the margin in increased (black bars in (G, H)). (I) WT embryo after ISH with eng2 and (I') with eng2 and otx2 (red). (J) gbx1-injected embryo; eng2 is not expressed ectopically. (J') Combined ISH with otx2 (red) shows that the remaining domain is located caudally to the reduced otx2 domain. (K) WT embryo after ISH with wnt1 and (K') with wnt1 and otx2 (red). (L) gbx1-injected embryo;wnt1 is not expressed ectopically. (L') In all gbx1-injected embryos the MHB is shifted anteriorly as indicated by green arrowheads. (M) WT embryo after ISH with hoxa1 and otx2 (red). (N) gbx1-injected embryo; the hoxa1 expression doamin is enlarged. (O) WT embryo after ISH with cdx4 and otx2 (red). (P) gbx1-injected embryo; the cdx4 expression domain is enlarged. (Q) WT embryo after ISH with val and krox20 (red). (R) gbx1-injected embryo; val expression is not affected: the krox20 expression domain corresponding to rh3 (green arrowhead) is enlarged, whereas rh5 is less affected. (A, B, E-P) Lateral views, anterior to the left; (C, D-Q, R) dorsal views, anterior to the top; (A'-L') flat-mounted embryos, anterior to the top.
Figure 3. gbx1 acts as a mediator of the Wnt8 hindbrain posteriorizing signal. (A) Control embryo at 24 h, (B) gbx1-injected embryo (200 pg), (C) wnt8-injected embryo (400 pg). gbx1 overexpression mimics wnt8 gain-of-function; the embryos are truncated anteriorly. The red arrowhead indicates the position of the ear. (D-G') Animal pole views at 60% epiboly after ISH with otx2. (D) Control embryo, (E, E') gbx1-injected embryos (200 pg), (F) embryo injected with wnt8 morpholinos, (G, G') embryos co-injected with gbx1 mRNA and wnt8 morpholinos. Expansion of the otx2 domain in the wnt8 morphant is rescued by co-injection with gbx1 mRNA. (H-K') Dorsal views at the tailbud (tb) stage after ISH with otx2 (blue) and krox20 (red). (H) Control embryo, (I, I') gbx1-injected embryos, (J) embryo injected with wnt8 morpholinos, (K, K') embryos co-injected with gbx1 mRNA and wnt8 morpholinos. Loss of krox20 and posterior expansion of the otx2 domain in the wnt8 morphant is rescued by gbx1 mRNA.
Figure 4. gbx1 induces posterior neural fate via transformation of anterior neural fate and gbx1 and otx2 repress each other cell-autonomously. (A) Schematic drawing of the fluorescein uncaging procedure (for details, see [33]). (B, C) gbx1-injected embryo after uncaging cells in the prospective otx2 domain. (D, E) After 20 h of development the labeled cells come to lie in the anterior of the embryo (arrow). (B, D) Nomarski optics; (C, E) detection of the fluorescent cells (arrow). (F) Experimental procedure for cell transplantations. Donor embryos are generated by co-injecting biotinylated dextran as lineage tracer and gbx1 (300 pg) or otx2 mRNA (300 pg). Cells are taken out of the donor at 40% epiboly and transplanted into a wild-type (WT) host embryo. (G) Chimeric embryo containing cells derived from embryos injected with biotinylated dextran and gbx1mRNA, and stained for otx2 (blue). Unlabeled patch of cells marked by a white arrow. (G') Close-up of the patch indicated in (G) before biotin staining and (G") after biotin staining (brown). gbx1 overexpressing cells within the otx2 domain do not express otx2. (H) Chimeric embryos containing cells derived from embryos injected with biotinylated dextran and otx2 mRNA, and stained for gbx1 (blue). Unlabeled patch of cells marked by a black arrow. (H') Close-up of the patch indicated in (H) before biotin staining and (H") after biotin staining (brown). otx2 overexpressing cells within the gbx1 domain do not express gbx1.
Figure 5. gbx1 morpholinos affect hindbrain patterning. (A-C') Embryos at the four somite stage stained for otx2/krox20/myod. (A) Control embryo, (B, B') embryos injected with 10 ng Mo1, (C, C') embryos injected with 7 ng Mo2. Injections of two different morpholinos show the same phenotype morphologically and after in situ hybridization analysis. (D-F) Rescue experiments using the krox20 expression domain as a read-out. (D) gbx1-morphant, (E, E') rescued morphant embryos co-injected with gbx1 mRNA that does not contain the MO binding sequence in its 5' region. (F) Two combinations were tested: 10 ng of Mo1 with 200 pg or 400 pg of gbx1 mRNA. (G, H) Western blot detection showing down regulation of gbx1-myc mRNA translation in embryos co-injected with gbx1-myc mRNA containing morpholino binding sequences and Mo1 (G) or Mo2 (H). WT, wild type.
Figure 6. Loss of gbx1 affects hindbrain and midbrain-hindbrain boundary (MHB) markers. (A-H, M-K-T) are dorsal views, (I, J) are lateral views and the developmental stages are indicated (tb, tailbud stage). (A) Control embryo (wild type (WT)) stained with val (blue) and krox20 (red), (B, B') gbx1-morphants. A residual krox20 expression is seen in (B') and no expression of krox20 in (B"). For (B', B") a higher magnification of the region of interest is shown. (C) Control embryo stained with hoxb1; (D) gbx1-morphant. (E) Control embryo stained with hoxa1; (F) gbx1-morphant. (G) Control embryo stained with hoxa1 and otx2; (H) gbx1-morphant. A higher magnification is shown, illustrating the reduction of size of the gap between the otx2 and hoxa1 domains. (I) Control embryo stained with cdx4; (J) gbx1 morphant. (K) Control embryo stained with pax2.1; (L) gbx1-morphant. The red bars show the distance between the MHB and the lateral plate mesoderm expression domains. This distance is markedly reduced in the gbx1 morphants. (M, O) Control embryos stained with otx2 (blue) and Ntl antibody (brown); (N, P) gbx1 morphants. The otx2 domain is unaffected at 60% epiboly but slightly shifted posteriorly at 70% (red arrowheads). (Q, S) Control embryos stained with fgf8; (R, T) gbx1-morphants. MHB expression is delayed (R) and shifted posteriorly (S, T, red arrowheads) in the morphants.
Figure 7. The otx2/gbx1 interface plays an essential role in midbrain-hindbrain boundary development. (A) Lateral view of a chimeric embryo stained for fgf8. The red arrowhead indicates ectopic fgf8 expression. (B) Lateral view of a chimeric embryo stained for pax2.1. The red arrowhead indicates ectopic pax2.1 expression. (C) Lateral view of a chimeric embryo stained for eng2. The red arrowhead indicates ectopic eng2 expression. (A', B', C') Close-up of the ectopic patch before biotin staining and (A", B", C") after biotin staining (brown). For all the three genes, ectopic expression is induced in gbx1-overexpressing cells within the otx2 domain (red arrowheads). (D) Summary table of the transplanted cells' positions and marker induction. Clones localized in the forebrain never induced ectopic fgf8, pax2.1 or eng2 expression.
Acampora,
Visceral endoderm-restricted translation of Otx1 mediates recovery of Otx2 requirements for specification of anterior neural plate and normal gastrulation.
1998, Pubmed
Acampora,
Visceral endoderm-restricted translation of Otx1 mediates recovery of Otx2 requirements for specification of anterior neural plate and normal gastrulation.
1998,
Pubmed
Acampora,
Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation.
1995,
Pubmed
Alexandre,
Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype.
1996,
Pubmed
Ang,
A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain.
1996,
Pubmed
Bellipanni,
Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos.
2000,
Pubmed
Bouillet,
Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonal carcinoma cells.
1995,
Pubmed
,
Xenbase
Broccoli,
The caudal limit of Otx2 expression positions the isthmic organizer.
1999,
Pubmed
Canning,
Sustained interactive Wnt and FGF signaling is required to maintain isthmic identity.
2007,
Pubmed
Crossley,
Midbrain development induced by FGF8 in the chick embryo.
1996,
Pubmed
Davidson,
cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes.
2003,
Pubmed
Ekker,
Coordinate embryonic expression of three zebrafish engrailed genes.
1992,
Pubmed
Erter,
Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo.
2001,
Pubmed
,
Xenbase
Fjose,
A zebrafish engrailed-like homeobox sequence expressed during embryogenesis.
1988,
Pubmed
Gamse,
Early anteroposterior division of the presumptive neurectoderm in Xenopus.
2001,
Pubmed
,
Xenbase
Garda,
Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer.
2001,
Pubmed
Hidalgo-Sánchez,
Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon.
1999,
Pubmed
Irving,
Regeneration of isthmic tissue is the result of a specific and direct interaction between rhombomere 1 and midbrain.
1999,
Pubmed
Joly,
Expression of a zebrafish caudal homeobox gene correlates with the establishment of posterior cell lineages at gastrulation.
1992,
Pubmed
Joyner,
Engrailed, Wnt and Pax genes regulate midbrain--hindbrain development.
1996,
Pubmed
Joyner,
Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer.
2000,
Pubmed
Katahira,
Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum.
2000,
Pubmed
Kelly,
Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways.
1995,
Pubmed
,
Xenbase
Kelly,
Expression of wnt10a in the central nervous system of developing zebrafish.
1993,
Pubmed
Kikuta,
gbx2 Homeobox gene is required for the maintenance of the isthmic region in the zebrafish embryonic brain.
2003,
Pubmed
Kozlowski,
Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.
1997,
Pubmed
Krauss,
Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis.
1991,
Pubmed
Lekven,
Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning.
2001,
Pubmed
Lekven,
Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary.
2003,
Pubmed
Li,
Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.
2001,
Pubmed
Liu,
Early anterior/posterior patterning of the midbrain and cerebellum.
2001,
Pubmed
Liu,
EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region.
2001,
Pubmed
Lun,
A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary.
1998,
Pubmed
Macdonald,
Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain.
1994,
Pubmed
Martinez,
Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en.
1991,
Pubmed
Martinez-Barbera,
Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2.
2001,
Pubmed
McGrew,
Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus.
1999,
Pubmed
,
Xenbase
Meinhardt,
Cell determination boundaries as organizing regions for secondary embryonic fields.
1983,
Pubmed
Mercier,
Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish.
1995,
Pubmed
Millet,
A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer.
1999,
Pubmed
Millet,
The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick/quail homotopic grafts.
1996,
Pubmed
Moens,
valentino: a zebrafish gene required for normal hindbrain segmentation.
1996,
Pubmed
Momoi,
Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8.
2003,
Pubmed
Morita,
Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain.
1995,
Pubmed
Nasevicius,
Effective targeted gene 'knockdown' in zebrafish.
2000,
Pubmed
Oxtoby,
Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development.
1993,
Pubmed
Prince,
Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino.
1998,
Pubmed
Raible,
Divide et Impera--the midbrain-hindbrain boundary and its organizer.
2004,
Pubmed
Reifers,
Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis.
1998,
Pubmed
Reim,
Spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development.
2002,
Pubmed
Rhinn,
Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling.
2005,
Pubmed
Rhinn,
The midbrain--hindbrain boundary organizer.
2001,
Pubmed
Rhinn,
Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification.
1998,
Pubmed
Rhinn,
Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling.
2003,
Pubmed
Rhinn,
Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain.
1999,
Pubmed
Rhinn,
Isolation and expression of the homeobox gene Gbx1 during mouse development.
2004,
Pubmed
Rupp,
Xenopus embryos regulate the nuclear localization of XMyoD.
1994,
Pubmed
,
Xenbase
Scholpp,
Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon.
2003,
Pubmed
Seo,
Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.
1998,
Pubmed
Shanmugalingam,
Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.
2000,
Pubmed
Shimizu,
Interaction of Wnt and caudal-related genes in zebrafish posterior body formation.
2005,
Pubmed
Simeone,
Positioning the isthmic organizer where Otx2 and Gbx2meet.
2000,
Pubmed
Simeone,
A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo.
1993,
Pubmed
Tour,
Otx2 can activate the isthmic organizer genetic network in the Xenopus embryo.
2002,
Pubmed
,
Xenbase
Tour,
Gbx2 interacts with Otx2 and patterns the anterior-posterior axis during gastrulation in Xenopus.
2002,
Pubmed
,
Xenbase
Wassarman,
Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function.
1997,
Pubmed
Wassef,
Early mesencephalon/metencephalon patterning and development of the cerebellum.
1997,
Pubmed
Waters,
Cloning and embryonic expression analysis of the mouse Gbx1 gene.
2003,
Pubmed
Weidinger,
The Sp1-related transcription factors sp5 and sp5-like act downstream of Wnt/beta-catenin signaling in mesoderm and neuroectoderm patterning.
2005,
Pubmed
Weinberg,
Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.
1996,
Pubmed
Woo,
Order and coherence in the fate map of the zebrafish nervous system.
1995,
Pubmed
Woo,
Specification of the hindbrain fate in the zebrafish.
1998,
Pubmed
Woo,
Specification of the zebrafish nervous system by nonaxial signals.
1997,
Pubmed
,
Xenbase
Wurst,
Neural plate patterning: upstream and downstream of the isthmic organizer.
2001,
Pubmed
