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First published online 26 November 2008
doi: 10.1242/dev.029157

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FGF3 in the floor plate directs notochord convergent extension in the Ciona tadpole

¹ Department of Molecular and Cell Biology, Center for Integrative Genomics, University of California, Berkeley, 142 LSA#3200, Berkeley, CA 94720, USA.
² Center for Cell Dynamics, Friday Harbor Laboratory, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA.

View larger version (67K): [in this window] [in a new window]   Fig. 1. FGFR is required for mediolateral convergence of the Ciona notochord. (A,B) Ci-FGFR is strongly expressed in the developing notochord plate (A, neurula stage, white circle) and in notochord after intercalation (B, tailbud stage). (C-D') Wild-type notochord cells (Bra::GFP, green; phalloidin, red) undergo progressive ML intercalation (C,C', early tailbud) to form a single cell row (D,D', late tailbud). (E,E') Noto1::dnFGFR-Venus-expressing (green) notochord cells display cell-autonomous defects in intercalation at early tailbud stage. (F,F') Noto1::dnFGFR+Bra::GFP (green) embryos display strong intercalation defects at late tailbud stage. (53/75 embryos displayed CE defects.) Images in C'-F' are magnifications from C-F, respectively. (G,H) dpErk staining of wild-type embryos at neurula (G) and late tailbud (H) stages shows absence of MAPK activation in the notochord. (I) Treatment of embryos with U0126 from late neurula stage on eliminates MAPK activation but does not affect notochord intercalation (103/103 embryos). (J) Distribution of the number of actin protrusions per internal notochord edge versus the ratio of ML- to AP-oriented protrusions. Note the difference between wild-type (blue) and dnFGFR (red) notochord indices (eight embryos each). P-values are indicated for each axis.

View larger version (75K): [in this window] [in a new window]   Fig. 2. Ci-FGF3 is expressed in the ventral midline of the nerve cord during notochord CE. (A) Ci-FGF3 in situ showing expression in the floor plate at mid-tailbud stage. (B,C) Cs-FGF3 enhancer::lacZ in situ in late neurula (B; inset, high magnification) and mid-tailbud (C) embryos recapitulates endogenous Ci-FGF3 expression. (D,E) Cross-sectional (D) and lateral (E) views of late neurula embryos double labeled for Cs-FGF3 enhancer (green, lacZ in situ) and Bra::GFP (red, GFP antibody). (F) Cross-sectional view of late neurula embryo stained with phalloidin (green). The notochord plate (dashed line) and floor plate precursors (asterisks) are indicated. (G) Cross-sectional illustration of the organization of major Ciona tissues: ventral nerve cord (red), lateral nerve cord (light blue), dorsal nerve cord (purple), notochord (green), tail muscle (dark blue), endoderm (yellow) and epidermis (brown).

View larger version (94K): [in this window] [in a new window]   Fig. 3. Ci-FGF3 is required for notochord CE. (A-C') Phenotypic series of embryos co-injected with Ci-FGF3 splice morpholinos (MOs) and Bra::GFP (green) and counterstained with phalloidin (red). The notochord displays progressively stronger intercalation defects ranging from an occasional two cells per row (A,A', category II), two-cell row (B,B', category III) and more than two cells per row (C,C', category IV). (D) Summary of MO phenotypes. n, number of embryos examined. (E) Distribution of actin protrusion abundance and ML/AP ratio in wild-type and MO embryos. The MO embryos display a more severe reduction in protrusions and loss of ML polarity than dnFGFR notochord (see Fig. 1J). (F-I') Misexpression of Ci-FGF3 disrupts notochord intercalation non-cell-autonomously. Notochord (F,F'), ventral and dorsal nerve cord (G,G'), muscle (H,H') and endodermal strand (I,I') enhancer-driven Ci-FGF3 causes varying degrees of intercalation defects (number of defective embryos/total embryos) in the notochord. All embryos are co-electroporated with Bra::GFP (green) and counterstained with phalloidin (red). Images in A'-I' are magnifications from A-I, respectively.

View larger version (102K): [in this window] [in a new window]   Fig. 4. FGF signaling is required for membrane localization of the PCP pathway proteins Dsh and PKC- in Xenopus dorsal mesoderm. Dsh-GFP and PKC--YFP proteins are membrane-localized in wild-type Xenopus dorsal marginal zone (DMZ) (A-B') and cytoplasmically localized in the animal cap (AC) (E-F'). Treatment with the FGFR inhibitor SU5402 between stages 10.5 and 12 causes Dsh and PKC- to delocalize from the membrane in DMZ (C-D'); compare with DMSO-treated controls (A-B'). All embryos are co-injected with membrane RFP (mRFP, red) to label the cell membrane.

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