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First published online 5 November 2003
doi: 10.1242/dev.00845

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FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors

¹ Howard Hughes Medical Institute, Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA
² Howard Hughes Medical Institute and Skirball Institute of Biomolecular Medicine, Departments of Cell Biology, and Physiology and Neuroscience, NYU School of Medicine, New York, NY 10016, USA

View larger version (77K): [in a new window]   Fig. 1. FGF8 begins to be expressed in the mouse mid/hindbrain region prior to Fgf18 and can induce Fgf18 expression in mouse midbrain explants. (A-C) Fgf8 is first expressed (arrowheads) in the mid/hindbrain region at the four-somite stage (C). (D-F) Fgf18 is first expressed in the mid/hindbrain region at the five-somite stage (arrowhead in E) and becomes restricted to a narrow transverse band straddling the isthmus by E9.5 (arrowhead in F; asterisk in D indicates the presumptive mid/hindbrain junction region). (G,H) Fgf18 is induced by FGF8b-soaked beads (arrowhead in H) by 48 hours, but not by BSA-soaked beads (G). Inset in H shows that Fgf18 is induced by FGF8b by 16 hours. (I,J) Fgf17 expression is first detectable in the mid/hindbrain region at the six-somite stage and at E9.5 it is in a broad domain on both sides of the mid/hindbrain junction (arrowheads). (K,L) Fgf17 is not induced after 48 hours by either the BSA-soaked or FGF8-soaked beads in rostral midbrain explants. Arrowhead in L indicates the endogenous Fgf17 expression sustained in the explant. Broken lines in F and J indicate the tissues used for the explant assays.

View larger version (56K): [in a new window]   Fig. 2. Spry1 is induced by FGF8b-soaked beads in prosomere 1 explants in the absence of protein synthesis. Spry1 (A) and Spry2 (B) are expressed in broad domains of the mid/hindbrain region at E9.5 Spry1 is induced by FGF8b-soaked beads within 4 hours of culture (D), but not by BSA-soaked beads (C). (E-H) Spry1 is induced within 6 hours by FGF8 in the presence or absence of 50 µg/ml cyclohexamide (CHX) in explants grown in medium containing 0.1% ethanol, but not by BSA-soaked beads. Red arrows in D,F,H indicate induced gene expression. Green circles in C and H indicate beads that were lost during processing of the tissues. Broken lines in A and B indicate the tissues used for the explant assays.

View larger version (66K): [in a new window]   Fig. 3. Fgfr2 and Fgfr3 expression is excluded from the mid/hindbrain junction and repressed by FGF8b. (A,B) Fgfr1 is weakly expressed throughout the embryo at the five-somite stage and E9.5, with the exception of the heart (h in B) at E9.5. (C,D) Fgfr2 is expressed in the brain at E8.5 and E9.5 in the forebrain, rostral midbrain and part of the posterior hindbrain (arrowheads), but excluded from the mid/hindbrain region (asterisks). (E,F) In the brain, Fgfr3 is expressed weakly in the caudal forebrain and part of the posterior hindbrain (arrowheads), but not in the mid/hindbrain region (asterisks) at the five-somite stage and E9.5. Note the strong expression in the extra-embryonic tissues at E8.5. (G,H) Weak and patchy Fgfr1 expression is seen in p1 explants after 48 hours and this expression is not altered by FGF8b-soaked beads. (I) In control p1 explants, strong Fgfr2 expression is limited to the dorsal midline, whereas weak expression is maintained in the rest of the explants. (J) Fgfr2 expression is downregulated by FGF8b-soaked beads. (K) Fgfr3 is maintained in BSA-treated p1 explants, whereas Fgfr3 is repressed by FGF8b-soaked beads (L). Broken lines in B,D,F indicate the tissues used for the explant assays.

View larger version (46K): [in a new window]   Fig. 4. FGF8b represses midbrain development whereas FGF8a, FGF17b and FGF18 promote midbrain development. (A) Schematic diagram showing the in ovo electroporation experiments. DNA (green) is injected into the midbrain by a glass needle and five electric pulses are applied. DNA is driven toward the anode and transfected only on the right side of the brain, whereas the left side serves as an internal control. (B) Co-transfection of a GFP expression vector with the experimental vector serves to show that most cells on the right side of the brain, including the mid/hindbrain region and caudal forebrain, are transfected. (C) Dorsal view of a wild-type E10.5 chicken brain. (D) Dorsal view of an E10.5 chicken brain electroporated with 1µg/µl pMiw-Fgf8b; the asterisk indicates lack of midbrain (mes) on the transfected right side. (E) Dorsal view of an E6.5 chicken brain electroporated with 1 µg/µl pMiw-Fgf8a; the midbrain on the transfected side is larger than the one on the control side. (F) Dorsal view of an E8.5 chicken brain electroporated with 1 µg/µl pMiw-Fgf17b; the midbrain on the transfected side is larger than the one on the control side. (G) Dorsal view of an E8.5 chicken brain electroporated with 1 µg/µl pMiw-Fgf18; the midbrain on the transfected side is larger than the one on the control side. tel, telencephalon; di, diencephlon; mes, mesencephalon; ce, cerebellum.

View larger version (115K): [in a new window]   Fig. 5. FGF8b activates more molecular pathways than FGF8a, FGF17b and FGF18. (A-D) 1 µg/µl pMiw-Fgf8a induces Fgf8 (C), but does not induce Gbx2 (B) or Spry1 (D), or repress Otx2 (A). (E-H) 1 µg/µl pMiw-Fgf8b induces Gbx2 (F), Fgf8 (G) and Spry1 (H) and represses Otx2 (E). (I-L) 1 µg/µl pMiw-Fgf17b fails to induce Gbx2 (J), Fgf8 (K) or Spry1 (L), or to repress Otx2 (I). (M-P) 1 µg/µl pMiw-Fgf18 fails to induce Gbx2 (N), Fgf8 (O) or Spry1 (P), or to repress Otx2 (M). (Q-T) 1 µg/µl pMiw-caFGFR2 induces Gbx2 (R), Fgf8 (S) and Spry1 (T) in scattered cells. Otx2 is repressed on the electroporated side but scattered Otx2-expressing cells still exist (Q). In all panels, coronal or near coronal sections are shown with the anterior end towards the right. The broken lines indicate the midline with the electroporated side above the line and the control side below. In all panels, the red arrowheads indicate ectopic gene expression on the electroporated side and the green arrowheads indicate endogenous expression on the control side except for E and Q where the red arrowheads indicate the electroporated side where Otx2 expression is repressed (completely in E and incompletely in Q). Insets in A,C,E,G,I,M,Q show expression of the mouse or human genes electroporated into the right side of the brain.

View larger version (38K): [in a new window]   Fig. 7. Activated FGFR1 and FGFR2 produce over-proliferation of the midbrain. (A) Dorsal view of an E10.5 chicken brain electroporated with 1 µg/µl pMiw-Fgfr1K656E, the midbrain on the transfected side is larger than the one on the control side. (B) Dorsal view of an E7.5 chicken brain electroporated with 3 µg/µl pMiw-Fgfr1N546K, the midbrain on the transfected side is larger than the one on the control side. (C) Dorsal view of an E7.5 chicken brain electroporated with 1 µg/µl pMiw-Fgfr2C342Y, the midbrain on the transfected side is larger than the one on the control side. In all panels, the right sides are the experimental sides and the left sides serve as controls. Broken outline indicates the expanded midbrain.

View larger version (100K): [in a new window]   Fig. 6. FGF8b and FGF17b proteins differentially regulate genes in mouse brain explants. Beads soaked in BSA, mouse FGF8b or human FGF17b, as indicated, were placed in midbrain explants and cultured for 48 hours. Wholemount RNA in situ analysis was then performed with the indicated probes. FGF8b strongly induces Gbx2 (E), Spry1 (H) and En1 (K), and represses Otx2 (B); FGF17b weakly induces En1 (L) and Spry1 (I).

View larger version (82K): [in a new window]   Fig. 8. FGF8b transforms the midbrain into a cerebellum and shifts the mid/hindbrain organizer rostrally. (A) Experiments shown in B, C and D. 24 hours after 1 µg/µl pMiw-Fgf8b was electroporated into the midbrain, Fgf8 expression is shifted into the caudal forebrain region on the experimental side, as well as in a thin band along the dorsal midline (red lines in A and arrowheads in B) that connects the ectopic Fgf8 domain to the endogenous Fgf8 domain on the control side. Green arrow shows the down regulation of Fgf8 expression on the transfected side in the isthmus. Inset shows the rear view of the same embryo. (C) 24 hours after 1 µg/µl pMiw-Fgf8b is electroporated into the chicken midbrain En1 expression is shifted rostrally on the electroporated side, and seen in the most dorsal cells in the midbrain and anterior hindbrain (red arrowheads), whereas the endogenous expression surrounding the isthmus (green arrow) is downregulated. Inset shows a rear view of the same embryo, note the normal expression on the control (left) side. (D) 24 hours after 1 µg/µl pMiw-Fgf8b is electroporated into the chicken midbrain, the endogenous Wnt1 expression in the isthmus (green arrow) is downregulated, whereas ectopic expression is induced near the dorsal midline and in a transverse band in the rostral midbrain. Inset shows a rear view of the same embryo, note the normal expression on the control (left) side. (E) Scattered expression of Fgf8 is induced in the midbrain and caudal forebrain (arrowheads) by ectopic expression of activated FGFR2. Note that the endogenous Fgf8 expression is not repressed. (F) Scattered expression of Wnt1 is induced in the midbrain and caudal forebrain (arrowheads) by ectopic expression of activated FGFR2. Inset shows a rear view of the same embryo.

View larger version (15K): [in a new window]   Fig. 9. FGF signaling is autoregulated at multiple levels and multiple FGF proteins regulate midbrain and cerebellum development. In the mouse, FGF8 expression in the isthmus at the four-somite stage represses the expression of Fgfr2 and Fgfr3 and activates the expression of Fgf18 at the five-somite stage. Fgf17 expression is initiated in a broader domain slightly later, and by E9 the three Fgfs are expressed in overlapping gradients radiating from the isthmus, whereas Fgfr2 and Fgfr3 are absent in this region. Spry1/2 genes are upregulated by FGFs. FGF8b is required to maintain a cascade of gene expression that includes absence of Otx2 in r1, allowing cerebellum development to occur. FGF17 and FGF18, and possibly FGF8a and a low level of FGF8b regulate growth and En expression in the midbrain.