Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary

Abstract

In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.

Fig. 1.

Analysis of Otx2 and EGFP expression, which mimics endogenous Gbx2, in the mes–r1 area of Gbx2^CreER/+ embryos. (A,B) EGFP fluorescence in Gbx2^CreER/+ embryos at E7.5 (A) and E8.5 (B). (C,D) Immunofluorescence of Otx2 and GFP on sagittal sections of Gbx2^CreER/+ embryos at E8.5 (C) and whole-mount embryos at E9.5 (D). Arrows mark the prospective MHB. (E-F′) Confocal images of Otx2/GFP immunofluorescence in flat-mount E9.5 neural tube segment indicated by dashed lines in D. The boxed area in E is magnified in F–F′. The MHB is marked by a dashed line; the arrow indicates the ventral midline; arrowheads mark Otx2⁺/GFP^– cells in r1 region. (G) Immunofluorescence of Otx2 and GFP on sagittal sections of Gbx2^CreER/+ embryos at E9.5 showing overlapping expression of EGFP and Otx2 in the ventral midline of the mes. The inset shows the relative position (boxed area) of G. (H-J) Immunofluorescence of Otx2 and TuJ1 on sagittal sections of Gbx2^CreER/+ embryos at E9.5 (H) and E10.5 (I,J). The inset shows the relative position of H in the dorsal mes-r1 junction (box). Otx2⁺ cells (arrowheads) in anterior r1 express TuJ1. The boxed area in I is enlarged in J. The arrowhead indicates Otx2⁺ cells in anterior 1.

Fig. 2.

Descendants of Gbx2-expressing cells are restricted posterior to the MHB. (A-C) X-gal histochemistry of Gbx2^CreER/+; R26R^lacZ/+ embryos at E8.5 (A) and E9.5 (B,C) following tamoxifen administration at E6.75. The arrowhead indicates the MHB. (D-F) X-gal histochemistry of whole-mount (D) and sagittal section of E10.5 Gbx2^CreER/+; R26R^lacZ/+ embryos (E), and horizontal section of E12.5 embryos (F) that received tamoxifen at E8.5. The inset in E shows in situ hybridization of Otx2 transcripts on sagittal section of E10.5 embryo. Note that X-gal⁺ cells are largely restricted to areas posterior to the caudal limit (dashed line) of the Otx2 expression domain, except for the ventral midline of the midbrain (arrow). The inset in F illustrates the plane of section (red line). (G,H) X-gal histochemistry (G) and in situ hybridization of Otx2 transcripts (H) on sagittal sections of Gbx2^CreER/+; R26R^lacZ/+ embryos at E18.5 after tamoxifen gavage at E10.5. cb, cerebellum; ic, inferior colliculus; ov, otic vesicle; sc, superior colliculus; tg, trigeminal ganglion.

Fig. 3.

Descendants of Gbx2-transcribing cells contribute to the midbrain in Gbx2-deficient mice. (A,B) X-gal histochemistry of whole-mount Gbx2^CreER/+; R26R^lacZ/+ (A) and Gbx2^CreER/–; R26R^lacZ/+ embryos (B) at E14.5 after tamoxifen administration at E6.75. (C,D) Immunofluorescence of Otx2 and YFP on sagittal sections of E12.5 Gbx2^CreER/+; R26R^YFP/+ (C) and Gbx2^CreER/–; R26R^YFP/+ embryos (D) given tamoxifen at E6.5. Note that the marked cells (asterisk) are present throughout the midbrain in Gbx2-deficient embryos, in contrast to the lineage restriction at the MHB (arrowhead) in the control embryos. The inset in D shows that the fate-mapped cells (YFP⁺) express Otx2. The bracket marks r1.

Fig. 4.

Gbx2-deficient cells ectopically express Otx2 and segregate from wild-type cells in the anterior hindbrain of chimeric embryos. (A-D) X-gal histochemistry of sections of chimeric embryos at E8.5 (A and B) and E16.5 (C and D). The inset in D is in situ hybridization of Otx2 transcripts on an adjacent section. (E-H) X-gal and in situ hybridization on adjacent horizontal sections of mutant chimeric embryos at the five-somite stage (E,F) and E10.5 (G,H). The planes of section are illustrated in the insets. The arrowheads indicate aggregates composed of either wild-type or mutant cells in r1; the arrow indicates the accumulation of mutant cells in the posterior mes; the dashed line marks the MHB boundary. F, forebrain; H, hindbrain; M, midbrain.

Fig. 5.

Different requirements for Fgf8 and Gbx2 in the maintenance of the lineage boundary at the MHB. (A-I) X-gal analysis of whole-mount (A,C,E), flat-mount (B,D,F) E10.5 brains, and whole-mount (G,H,I) E12.5 brains of indicated genotypes. Tamoxifen was given at E6.75. The neural tube segment used for flat-mount preparations is demarcated with red lines. The dashed line and arrows mark the isthmus. Insets show the dorsal view of the embryos in G and I, and the coronal section of the embryo in H. The dashed line in H indicates the plane of section. Note that the neuroepithelium corresponding to r1 is enlarged (bracket) and abnormally folded. (J,K) X-gal histochemistry on sagittal section of Gbx2^CreER/F; R26R^YFP/lacZ (J) and Gbx2^CreER/+; Fgf8^F/–; R26R^lacZ/+ (K) embryos at E12.5 following tamoxifen exposure at E6.75. The inset in J shows double immunofluorescence of Otx2 and GFP on the adjacent section of J. Note that the anterior border of the fate-mapped cells is in the posterior mes (J), and that the cerebellar primordium was noticeably smaller, caused by the mosaic deletion of Fgf8 (K). (L-L′) Double immunofluorescence for Otx2 and GFP on the sagittal section of E10.5 Gbx2^CreER/+; Fgf82^F/–; R26R^YFP/+ that received tamoxifen at E6.75. The image corresponds to the dorsal part of the MHB, as indicated by the bracket in K. The asterisk indicates the fate-mapped cells in the mes.

Fig. 6.

Mosaic deletion of Gbx2 or Fgf8 results in distinct responses in gene expression in the mes-r1 area. (A-L) In situ hybridization of whole-mount embryos at E9.5 and E10.5 (insets). Genotypes and probes are indicated at the top and left, respectively. Arrowheads indicate ectopic expression in patches of r1 cells; arrows indicate the loss of expression of Fgf8 and Gbx2 or the ectopic expression of Wnt1 near the dorsal midline; black brackets in B, E and H mark the expanded expression domain of Gbx2, Fgf8 and Wnt1, respectively; red brackets in the insets of J and L demarcate the r1 domain; the asterisk indicates the loss of the transverse band of Wnt1 expression at the MHB.

Fig. 7.

Activation of FGF signaling has cell-autonomous function in cell sorting in the mes. (A,B) Distribution of cells transfected with EGFP alone (A) or EGFP with activated FGFR1 (B) in chick embryos at 48 hours after electroporation. (C-L′) Double fluorescence of GFP and Otx2 on horizontal sections of embryos transfected with EGFP (C,G-G′), activated FGFR1 (D and H-H′), Otx2 (E, I-I′,K-K′) and Otx2 with activated FGFR1 (F,J-J′,L-L′). Boxed areas in C, D, E and F are enlarged and shown in G-L′ as separate and merged color panels as indicated.