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spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development

Gerlinde Reim and Michael Brand*

Max Planck Institute for Molecular, Cell Biology and Genetics, Dresden, Pfotenhauer Str. 108, 01307 Dresden, FR of Germany



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  		Fig. 1. Brain phenotype of spg-embryos at pharyngula stages. (A,C,E,G,I) Wild-type and (B,D,F,H,J) homozygous spg mutant embryos. (A,B,E,F and small pictures in G,H) dorsal views; (C,D,G,H,I,J) lateral views. In the wild-type embryo (I), the MHB is marked by an arrow; The asterisk in B and the arrow in J indicate lack of the MHB in mutant embryos. (D,F) An arrowhead indicates a likely rudimentary tissue of the posterior cell row and the cerebellum after 28 hpf. (A,B) Phenotype of living embryos. (C-F) Optical sections of living embryos stained with fluorescent Bodipy-Ceramide. (G,H) Fluorescent staining with Acridine Orange indicates cell death at the prospective MHB and the optic stalk in spg embryos (H) at the 17-somite stage, indicated by arrows. Cell death is also detected in two transverse bands within the rhombencephalon at the 22-somite stage (arrowheads, insert in H; the arrow points to the MHB). (I,J) Sagittal histological sections.

 


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  		Fig. 2. The primordia of the MHB and the hindbrain affected in spg embryos. (A-B',E-F', I-J',N-N',Q,Q',U-U') Dorsal views; embryos in the remaining pictures are shown from lateral view. Gene expression, stages and genotypes are noted. Red arrowheads indicate expression of genes at the MHB throughout. (A-D') In spg embryos, pax2.1 shows reduced expression at the MHB from its onset onwards (A',B'), is lost during midsomitogenesis (C') and re-expressed as a dorsal patch after 24 hpf. Expression of pax2.1 within the otic placode (B-D') is not affected in mutant embryos. (E-H') wnt1 is normally expressed at its onset at 80% of epiboly (not shown), but becomes subsequently downregulated in mutant (E') at the time when pax2.1 is initiated. During somitogenesis (F-G'), the expression of wnt1 at the MHB (arrowhead) and within rhombomeres is downregulated in spg embryos. At pharyngula stages, wnt1 expression is continued within a dorsal patch at the MHB. (H,H') The midsagittal expression in the diencephalon seems unaffected, but MHB expression is reduced to a dorsal patch in mutant embryos. (I-I') fgf8 expression, like that of wnt1, is not affected in spg embryos at its onset of expression at the MHB (not shown), but soon becomes downregulated at around 90% of epiboly. (J) fgf8 expression caudally continues in r1, r2 and r4 in wild-type embryos. (J') In mutant embryos, fgf8 expression is strongly reduced within r1 and abolished within r2 and r4. During somitogenesis, fgf8 expression is completely lost from the MHB but, like pax2.1 and wnt1, recovers at a dorsal patch at the MHB. (M-P') spry4 is not properly initiated in spg embryos. At the four-somite stage, spry4 is strongly reduced at the MHB and in r1, r2 and r4 (N'). MHB expression of spry4 during somitogenesis and pharyngula stages follows the same mode as fgf8 and pax2.1. (Q,Q') en2 is normally initiated at the MHB at the end of gastrulation. In spg mutant embryos, en2 is downregulated from its initiation of expression. (R) en2 is expressed in the prospective tectum in a graded fashion during somitogenesis but is strongly reduced in spg embryos (R'). (S) en3 is encompassed within the en2 domain at the tectum in wild-type embryos. (S') In spg embryos, en3 is downregulated in a similar fashion as en2. (T,T') half sides of transverse sections through the her5 positive domain at the spatial level of the future MHB; arrows point to the neuroectoderm. (T) her5 is normally initiated within the neuroectoderm around 70% of epiboly, overlying mesendodermal expression. (T') her5 is not properly initiated in spg embryos. (U,U') her5 expression at the MHB is reduced in mutant embryos at the end of gastrulation.

 


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  		Fig. 3. Marker gene expresison at the MHB in spg.

 


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  		Fig. 4. Prosencephalic markers expand posteriorly in spg embryos. (A) emx1 is expressed in telencephalic precursors from end of gastrulation onwards in wild-type embryos. The posterior transverse expression domain marks the di-mesencephalic boundary. pax2.1 expression at the anterior MHB is shown in red. (A') In spg embryos, defined by the impaired expression of pax2.1 at the MHB, emx1 expression is generally elevated but reduced in its spatial lateral extent. The posterior border of emx1 expands caudally. (B) anf1, like emx1, is expressed at the anterior neural border with a patch of expression centering around the midline of the neuroectoderm. gbx2 expression at the posterior MHB is shown in red. (B') anf1 is lost within the midline expression domain in spg embryos, defined by impaired gbx2 expression (see Fig. 3 for gbx2 expression). (C) pax6 is initiated within the forebrain at the end of gastrulation. pou2 expression at the MHB is seen in red. (C') spg embryos, identified by loss of pou2 expression, show a posterior expansion of pax6 expression into the territory of the prospective MHB. (D,D') Double in situ hybridization with fgfr3 (blue) and en3 (red) at the 10-somite stage show the hindbrain domain of fgfr3 (arrow in D,D') is fused with the diencephalic domain of fgfr3, particularly at its ventral aspect. The MHB marker en3 is restricted to a dorsal patch in mutant embryos (D'). (E) During somitogenesis, besides its expression in the forebrain, pax6 is also expressed within the hindbrain and spinal cord in wild-type embryos. (E') The prosencephalic and the rhombencephalic domain nearly fuse in spg mutant embryos mainly owing to strong posterior expansion of the posterior border of the prosencephalic domain of pax6. (F,G) Anterograde filling of whole eyes with DiI (green fluorescence) or DiO (red fluorescence) shows a proper contralateral retinotectal mapping of RGC axons in spg embryos (F). The chiasma opticum is properly formed in spg mutant embryos (G).

 


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  		Fig. 5. The hindbrain primordium is affected in spg embryos. Embryos are photographed from the dorsal side, with the exception of D,D' (transverse sections at the level indicated by an arrow in C,C') and E, E' (lateral views). Dorsal is upwards in D,D'. Anterior is towards the top in A-C',F,F',R,R',S,S'; anterior is to the left in the remaining pictures. Embryos are at the tailbud stage unless indicated differently. (A) gbx1 expression is strictly posteriorly adjacent to the MHB domain of pax2.1 in wild-type embryos during gastrulation. (A') In spg embryos, pax2.1 and gbx1 are expressed in the same mutually exclusive fashion as seen in wild-type embryos at the end of gastrulation. However, in mutant embryos pax2.1 expression is reduced at the MHB. (B) In wild-type embryos, otx2 expression partially overlaps pax2.1 expression at the MHB at the end of gastrulation. (B') spg embryos show a proper spatial relationship of otx1 and pax2.1 at the prospective MHB at the end of gastrulation. (C,D) In wild-type embryos, gbx2 becomes activated at around 90% of epiboly within the neural ectoderm, shortly after onset in the underlying mesendoderm. (C',D') In mutant embryos, the mesendodermal domain of gbx2 is initiated normally (red arrowhead in D') but the neurectodermal domain of gbx2 is not initiated. Two longitudinal stripes in the non-neural ectoderm are unaffected (black arrow in D'). (E,E') In contrast to spg mutant embryos, gbx2 is lost in both the mesendodermal and the neuroectodermal germ layer in ace mutant embryos. (F) The hindbrain domain of fkd3 is lost in mutant embryos (F'). (G) In wild-type embryos, krox20 stains r3 and r5, and six3 is expressed within the prosencephalon, including the prospective eye field. (G') In mutant embryos, six3 seems not affected but krox20 is strongly reduced. (H) ephA4 is expressed in wild-type embryos within the prosencephalon and the rhombencephalon, in particular within rhombomeres 1, 3 and 5. (H') Rhombomeric expression of ephA4 is strongly affected and the prosencephalic domain shows massive posterior expansion. (I) wnt8b is normally expressed within the diencephalon, at the MHB and within rhombomeres 1, 3 and 5. (I') In spg embryos, MHB expression of wnt8b is strongly reduced (arrowhead) and rhombomeric expression is strongly downregulated; in particular, r1 cannot be discriminated from and possibly fuses with the MHB domain. (J) Double in situ staining for hoxb1a, expressed in r4, and hoxb4a, expressed within the spinal cord with an anterior limit at the border between r6 and 7 in wild-type embryos. The bracket indicates the gap between r4 and 7. (J') In mutant embryos, the gap between r4 and r7, indicated by the bracket, is strongly reduced. (K,K',L) pou2 expression becomes refined during early somitogenesis within distinct bilateral clusters, according to r2 and r4, and to a patch of expression at the posterior border of the MHB. (L') In early somitogenesis, embryos of the allele spge713 show strongly reduced rhombomeric expression of pou2, whereas in embryos carrying the insertional allele spghi349, pou2 expression is totally abolished (L''). (M) val is normally expressed within r5 and 6. (M') In mutant embryos, val expression is nearly abolished in r5 but the expression in r6 is not affected. val is also expressed within precursor cells of the neural crest (indicated by arrows in M,M'), which is not affected in mutant embryos. (N) fkd3 is expressed at inter-rhombomeric borders at late somitogenesis stages in wild-type embryos. (N') In spg embryos, inter-rhombomeric expression is strongly reduced. (O) zath1 is normally expressed at the prospective cerebellum and along the dorsal rim of the fourth ventricle. This expression is also maintained during later pharyngula stages (P). (O') zath1 expression is lost from the cerebellar anlage in spg embryos (arrow) but expression recovers partially at later stages (arrow in P'). (Q) In wild-type embryos, expression of otx2 at pharyngula stages covers the midbrain and the MHB, in particular the concise stripe of the posterior cell row (arrow) marking the transition between the tectum and the cerebellar anlage. (Q') In spg embryos, expression of otx2 partially recovers within this particular posterior cell row (arrow) at late pharyngula stages. The spatial extent of the midbrain territory of otx2 is apparently smaller than in wild-type embryos. (R) Among the proneural genes, ngn1 is expressed in precursors of primary neurons in wild-type embryos at the beginning of somitogenesis. (R') ngn1 is strongly abrogated in mutant embryos. (S) sox17 is normally expressed within the endodermal precursors in a punctate pattern during gastrulation (inset: transversal section at 70% epiboly, showing pou2 expression restricted to the neuroectodermal layer). (S') sox17 expression is strongly affected in mutant embryos. (T) myod is expressed within the paraxial mesoderm and muscle precursors within somites during somitogenesis. (T') myod expression is strongly reduced in the somitic mesoderm of spg embryos but the paraxial domain seems unaffected.

 


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  		Fig. 6. spg/pou2 requirement in the neuroectoderm. (A) Transplanted wild-type cells (brown) in spg embryos express gbx2 cell autonomously. All blue cells carry the brown transplantation marker. The right half of the embryo serves as a control: it is devoid of wild-type cells. As is normally seen in spg mutants (compare with Fig. 5D), gbx2 expression is found only in the mesendoderm, but not the overlying neuroectoderm. Dorsal view of a spg chimera, anterior is upwards. The white line indicates the plane of the transversal section in B along the gbx2 domain. (B) Cross-section of the embryo in A showing that the transplanted wild-type cells expressing gbx2 (bracket; arrow indicates the unaffected non-neural ectoderm domain, see also Fig. 5D') are located in the neuroectoderm. Other cells that are only brown lie outside the normal domain of gbx2 expression. (C,D) Transplanted wild-type cells (brown) in spg embryos also express pax2.1 normally at the MHB. Arrows point to the residual pax2.1 expression at the MHB which is retained in spg embryos until late stages of somitogenesis. (E) Clones of wild-type cells within the mesoderm cannot restore gbx2 expression in spg mutant embryos at the tailbud stage. The plane of section is similar to B. Arrows point to the unaffected non-neural ectoderm domain. (F) Mouse Oct3/Oct4/Pou5f1 is globally expressed within the neural plate at day 8.0 p.c. (dorsal view, anterior to the left). (G) spg/pou2 might be required to activate Fgf8-dependent gbx2 expression either for a planar or vertical signal. The transplantation experiments presented here show a requirement in the neuroectoderm. (H) Mouse Oct3/Oct4 mRNA and lacZ mRNA were co-injected into one cell of a two-cell stage zebrafish embryo. pax2.1 expression can be restored in spg mutant embryos by mouse Oct3/Oct4 mRNA (arrow, lacZ expression is indicated by the brown color) (Burgess et al., 2002Go). nec, neuroectoderm; mes, mesendoderm; tb, tailbud stage.

 


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  		Fig. 7. Relationship between pou2 and fgf8. All embryos are depicted dorsally with the exception of the embryo in F, which is depicted laterally. (A-D) Gain of fgf8 function by unilateral misexpression of fgf8 mRNA into one cell of two-cell stage embryos. To determine the effect caused by fgf8 overexpression, gbx2 (A,B) and spry4 (C,D), both markers for the prospective hindbrain, were used. The activity of misexpressed fgf8 can be judged from dorsalization of the embryos indicated by lateral expansion of endogenous gbx2 and spry4 expression, indicated by arrows (A-E) at the injected side of the embryo. Deposition of co-injected lacZ mRNA is visualized by staining for anti-ß-gal antibody (brown), reflecting the location of injected fgf8 mRNA (not visualized). Distribution of injected mRNA is restricted to one half of the embryo, allowing for comparison with the contralateral side as a control. In spg embryos, neither expression of gbx2 (B) nor spry4 (D) could be rescued or upregulated, respectively, by fgf8 overexpression. (E,F) In a reversed experiment, pou2 mis-expression into ace embryos (carried out in the same unilateral fashion described for fgf8 injection above), pou2 overexpression and fgf8 itself can provoke dorsalization of the injected half of the embryo (obviously seen in the wild-type embryo in E, but not in the ace embryo in F, owing to complete loss of the readout marker gbx2) but cannot rescue expression of gbx2 in ace mutant embryos. (H) A bead soaked with Fgf8 protein can not rescue the morphology of the isthmic constriction at the MHB but can evoke ectopic spry4 expression in wild-type and spg embryos (G,H; white circles indicate the implanted bead).

 


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  		Fig. 8. The double mutant spg-ace shows a more severe brain phenotype (J, living embryo) than each mutant alone (D, G, living embryos). (B,C,E,F,H,I,K,L,M-P) pax2.1 expression. At midsomitogenesis, the MHB expression of pax2.1 is severely reduced and completely missing at pharyngula stages in the double mutant embryos (K,L). (M-P) Phenocopy of the double mutant phenotype produced by blocking Fgf receptors using the inhibitor SU5402. (N,P) spg embryos treated with the inhibitor (+ SU5402, P) reveal strong similarity to spg/ace double mutant embryos, which is reflected by pax2.1 staining (compare P with K; expression of pax2.1 within the otic placode is also strongly reduced by inhibition (S. Léger and M. B., unpublished). (Q) During the first steps of regionalization of the MHB and the hindbrain, positioning of the MHB is independent of Pou2 (a). During the establishment phase of the MHB organizer, Pou2 is upstream of several cognate MHB markers (b). In the hindbrain primordium, spg/pou2 and ace/fgf8 serve a combinatorial role in initiation of gbx2 and spry4.

 

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© The Company of Biologists Ltd 2002