Gbx1 and Gbx2 function redundantly in cerebellum development. (A) Schematic of nonsense mutations identified in zebrafish gbx1 and gbx2 by TILLING. Both mutations are expected to prevent DNA binding by truncating the homeodomain. (B–I) Dorsal views at 22 hpf (B–E) or 6 dpf (F–I), anterior to the left. Genotypes are shown at the top. otx2 (blue), eng2b (red), and egr2b (blue) are expressed in midbrain, midbrain–hindbrain boundary (MHB), and rhombmere 3/5, respectively, in wildtype (WT), single and double mutants as shown. Tg(ptf1a:EGFP) (green) marks Purkinje neuron progenitors, Tg(olig2:DsRed2) (red) marks projection neurons, and anti-Vglut1/Slc17a7 (blue) marks cerebellar granule neuron axons. In gbx1^fh271;gbx2^fh253 double mutants the midbrain is expanded at the expense of r1 and no cerebellum forms.

Cerebellar development is rescued in gbx-embryos with otx knock-down. Dorsal views at 5 dpf, anterior is to the left. Genotypes are indicated at the top. (A–H) Zebrin II/Aldoca (A–D) and Vglut1/Slc17a7 (E–H) are expressed in cerebellar Purkinje cells and granule cell axons respectively; both are absent in gbx- and rescued in otxMO and gbx-;otxMO.

Rescue of cerebellar primordium morphogenesis in gbx-embryos with otx knock-down. Timelapse of embryos during the initial stages of cerebellar morphogenesis. Dorsal views with anterior to the left; genotypes are indicated at the top. Arrowheads indicate the isthmic constriction. (A–E) In WT embryos, inflation of the midbrain (III) and hindbrain (IV) ventricles combined with cell shape changes at the MHB creates a sharp isthmic constriction flanked posteriorly by a thickened bilateral cerebellar primordium (CbP, indicated on the left side with dotted white lines and on the right side by a white bracket indicating thickness). Red lines indicate a 90° rotation of the CbP relative to the more posterior hindbrain epithelium. (H–L) Ventricle inflation is delayed in gbx mutants, the isthmic region is short and no thickened CbP forms. (O–S; V–Z) In otxMO and gbx-;otxMO the isthmic region is extended and a thickened CbP forms. (F, M, T, and AA) The “isthmic region” is where the left and right sides of the neuroepithelium are in contact (white line), and can be easily measured at 22 hpf in WT (F). This region is nearly absent in gbx- (M), but extended in otxMO (T) and gbx-;otxMO (AA). (G, N, U, and BB) atoh1a expression in granule cell progenitors in the upper rhombic lip (URL, black arrows) is lost in gbx- (N) and rescued in both otxMO (U) and gbx-;otxMO (BB).

Rescue of the MHB program in gbx-embryos with otx knock-down. RNA in situ hybridization with genes shown on left; genotypes indicated at the top. Dorsal views of 22 hpf embryos with anterior to the left. (A–T) fgf8a, il17rd/sef, gbx2, pax2a and eng1b are all expressed at or around the MHB, are absent or strongly reduced in gbx- but are expanded in both otxMO and gbx-;otxMO embryos. (U–X) wnt1 is normally expressed in a narrow domain anterior to the MHB (U). This expression is reduced in gbx- (V) but rescued anterior to the extended isthmic region in otxMO (W) and gbx-;otxMO (X).

Changing requirements for wnt1 and fgf8 expression leads to rescue of a wnt1–fgf8 boundary in gbx-;otxMO embryos. RNA in situ hybridizations with wnt1 (red) and fgf8a (blue) at the stages shown on the left; genotypes indicated at the top. Dorsal views with anterior to the left. (A–D, F–I, and K–N) At 10.33 hpf (1–3 somites) in WT (A), wnt1 (red) and fgf8a (blue) are expressed in broad domains anterior and posterior to the presumptive MHB respectively (black arrow indicates the fgf8a domain in the anterior hindbrain). fgf8a is also expressed in hindbrain rhombomere 4 (black dot). In gbx-embryos wnt1 is initially expanded (B and G) but subsequently lost (L). Conversely, in otxMO and gbx-;otxMO, wnt1 expression is initially reduced (C and D) but subsequently recovers while the fgf8a domain expands (H, I, M, and N). (E, J, and O) Genetic pathways indicate the decreasing dependence of Wnt1 expression on Otx in the midbrain and its increasing dependence on Fgf8 signaling from r1.

Fgf signaling is required for rescue of the MHB program and cerebellar differentiation but not URL specification in gbx-;otxMO. Dorsal views of 22 hpf embryos (A–H) or 4 dpf larvae (I–L) with anterior to the left. Genotypes are indicated at the top. (A–H) RNA in situ hybridizations with genes indicated on left. atoh1a expression in the URL (black arrows in A) is absent in dnFgfr (B) but is rescued in dnFgfr;otxMO (C) and gbx-;dnFgfr;otxMO embryos (D). By contrast, MHB gene expression (pax2a, E) is not rescued by otx knock-down in the absence of Fgf signaling (G and H). (I–L) Zebrin II/Aldoca (red) normally expresses in cerebellar Purkinje cells and Tg(ptf1a:EGFP) (green) marks Purkinje neuron progenitors in WT (I; 14 larvae examined), but zebrin expression is absent and Tg(ptf1a:EGFP) is only expressed in lower rhombic lip in gbx-;dnFgfr;otxMO (L; 8 larvae examined). In dnFgfr (J) larvae, zebrin expression is absent (9 out of 18 larvae) or few zebrin-expressing cells are lying laterally at the junction of the tectum and the ptf1a:EGFP-expressing lower rhombic lip (the box in J; 9 out of 18). Similar to dnFgfr larvae, zebrin expression is either in the lateral junction between the tectum and lower rhombic lip in dnFgfr;otxMO (K; 15 out of 30 larvae) or is absent (the box in K).

MHB gene expression in gbx-, otxMO, and gbx-;otxMO embryos.
RNA in situ hybridizations with the genes and developmental stages indicated on the left. Genotypes are indicated at the top. Embryos are mounted in dorsal view with anterior to the left. (A-D) efnb2a is expressed in forebrain, r1, r4, and r7 in WT (A). The r1 expression of efnb2a is greatly reduced in gbx- (B), but expanded in both otxMO (C) and gbx-;otxMO (D). (E-H) otx2 is expressed in the midbrain of WT embryos (E), is expanded in gbx- (F) and shortened in otxMO (G). Otx is expressed throughout the isthmic region in gbx-;otxMO embryos (H) but is not functional due to the presence of the MOs. (I-L) eng1a is expressed at MHB in WT and otxMO (I,K), but is absent in gbx- and gbx-;otxMO embryos (J,L).

Single color in situs in support of Fig. 5.
RNA in situ hybridizations with wnt1, fgf8a, and pax2a at the stages shown on the left; genotypes indicated at the top. Dorsal views with anterior to the left. (A-L) 10.33 hpf embryos (1-3 somite stage). wnt1 (A-D) is expressed in the presumptive midbrain and is reduced in otxMO and gbx-;otxMO. fgf8a (EH) is expressed in presumptive r1 (black arrows) and r4 (black dots). pax2a (I-L) is expressed in a broad domain encompassing the MHB. The onset of fgf8a and pax2a expression is largely unaffected in mutants compared to WT. (M-T) At 14 hpf, wnt1 expression is still expanded in gbx- and reduced in otxMO and gbx-;otxMO (N-P), while fgf8a expression is progressively expanding in otxMO and gbx-;otxMO (S,T).

Fgf signaling is required for rescue of the MHB program but not URL specification in gbx- ;otxMO embryos; data in support of Fig. 6.
RNA in situ hybridizations at 22 hpf with the genes indicated on the left (A-H), and Bodipy staining at 2 dpf (I-L). Genotypes are indicated at the top. Embryos are mounted in dorsal view with anterior to the left. (A-D) atoh1a in the URL is absent in fgf8MO (B), but is rescued in fgf8MO;otxMO (C) and even more extensively rescued in gbx-;fgf8MO;otxMO (D). The midbrain (III) and hindbrain (IV) ventricles inflate without forming a sharp isthmic constriction when Fgf8 is knocked down. (E-H) fgf8a expression in anterior r1 is lost in fgf8a morphants (F) and is not rescued by Otx knock-down (G,H). (I-L) 2 dpf embryos stained with CellTraceTM Bodipy, imaged live. Doted lines in WT (I) show the cerebellar primordium (CbP). MHB morphogenesis is disrupted (the III and IV ventricles fuse into a single ventricle) in fgf8MO (J), and is not rescued by knock-down of Otx in otxMO (K) or gbx-;otxMO (L) in spite of prior rescue of cerebellar primordium morphogenesis and URL specification.