Expression pattern of tbx-c in the zebrafish embryo. (A) Dorsal view of a flat-mounted zebrafish embryo at 10 hpf. Note the expression in the anterior ventral neural keel, single eye field, the otic vesicle and the early notochord. (B) Transverse section of the 10 hpf embryo showing tbx-c transcripts in the eye field as well as in the anterior ventral neural keel. (C) Dorsal view showing expression of tbx-c at 20 hpf. The expression is localized to the ventral forebrain, the dorsal eye, the epiphysis, the trigeminal as well as lateral line ganglia and the otic vesicle. (D) Dorsal view of whole-mounted embryo (22 hpf) showing expression in pectoral fin buds and the otic vesicle. (E) Transverse section at the eye level shows tbx-c transcripts in the epiphysis, the ventral diencephalon and retinal cells in the dorsal eye. (F) Transverse section shows expression of tbx-c in the otic vesicle. (G) Transverse section through the caudal spinal cord shows expression of tbx-c in mechanosensory Rohon-Beard cells and in the excretory system. e, eye; ep, epiphysis; ex, excretory system; llg, lateral line ganglia; n, notochord; ov, otic vesicle; rb, Rohon-Beard cells; tg, trigeminal ganglion; vdc, ventral diencephalon; vnk, ventral neural keel. Anterior is at the left, unless otherwise stated. Scale bars, 100 μm (A), 50 μm (B-G).

Expression pattern of tbx-c, tbx-b and tbx-a in the tail and discrete cell groups in the chordoneural hinge (CNH) of zebrafish at 18-20 hpf. (A) tbx-c is expressed in the RB cells, the excretory system and the CNH (outlined). (B) tbx-b is expressed in the notochord. (C) tbx-a is expressed in the excretory system. (D) Double staining for Ntl protein (brown) and tbx-c mRNA (blue). Ntl is expressed in the posterior mesoderm, the CNH and the notochord. tbx-c transcripts map to the anterior CNH. (E) shh is expressed in the CNH, the notochord and the floor plate. (F) Two-colour in situ hybridization staining for tbx-c (blue) and flh (magenta). tbx-c is expressed in the anterior CNH, whereas flh is expressed in the posterior part of the CNH. Expression domains of flh and tbx-c overlap in the mid-CNH (arrows). (G) tbx-c is expressed in the anterior part of the CNH. (H) A model summarizing the distribution of gene products in the CNH and the notochord. cnh, chordoneural hinge; ex, excretory system; fp, floor plate; n, notochord; pm, posterior mesoderm. Scale bars, 25 μm

Effects of ectopic expression of tbx-c+ and dn-tbx-c in zebrafish embryos at 10 hpf. For each marker, control embryos injected with lacZ mRNA, tbx-c+ embryos and dn-tbx-c embryos were included. (A-H) Expansion of the notochord in tbx-c+ embryos (B,E) and attenuation of the notochord in dn-tbx-c embryos (C,F) are shown by expression of ntl and shh. Dorsal view of a flat-mounted control (G) as well as tbx-c+ (H) embryos show that overexpression of tbx-c+ causes bifurcation of anterior midline (H). (I-K) tbx6 expression in the lateral mesoderm (I) is reduced in tbx-c+ embryos (J) and expanded to the midline in dn-tbx-c embryos (K). (L-N) spt expression (L) like tbx6, is reduced in the lateral mesoderm of tbx-c+ embryos (M) and expanded in dn-tbx-c embryos (N). (OT) myoD is expressed in adaxial (arrow) and paraxial (arrowheads) cells (O). Its expression reveals a reduction of somitic tissue in tbx-c+ embryos (P,S) and fusion of somites at the midline in dn-tbx-c embryos (Q,T). Transverse sections reveal that the midline is expanded about 2.5-fold in tbx-c+ embryos (P,S) in comparison to that of the control (O,R). In contrast, the midline mesoderm is reduced and myoD-positive muscle cells appear in the midline of dn-tbx-c embryos (arrows; T). Scale bars, 100 μm (A-Q), 25 μm (R-T).

Double localization of the ntl and the Islet-1 reveals concurrent changes in the notochord and MNs of tbx-c+ and dn-tbx-c embryos at 11 hpf. The notochord is detected by ntl expression (blue) and neurons by the expression of Islet-1 (brown). (A,D) Control embryos featuring two bilateral lines of MNs along the midline and Rohon-Beard cells along the lateral neural plate. (B) Multiple notochords and extranumerary Islet-1-positive neurons in an embryo injected with a high dose of tbx-c+ mRNA. (C) Reduced notochord and a decreased number of MNs (arrow) in the dn-tbx-c embryo. (E) Extranumerary MNs with an expanded notochord in the tbx-c+ embryos. (F) The dn-tbx-c embryo showing a reduced number of MNs (arrow) and undetectable notochord. (A-C) Whole-mount embryos; (D-F) dorsal view of flat-mount embryos. mn, motor neurons; n, notochord; rb, Rohon-Beard cells. Scale bars, 100 μm (A-C); 25 μm (D-F).

Analysis of tbx-c expression and effect of ectopic expression of tbx-c+ in mutants. (A,B) Expression of tbx-c in wild-type (A) and ntl^-/- mutant (B) embryos. Note the segregation of axial mesoderm (arrowheads) and a low level expression of tbx-c in the midline at the trunk of the mutant (arrows). (C-E) Co-localization of ehh (blue) and Ntl (brown) in wild-type (C), tbx-c+ injected ntl^-/- (D) and normal sibling(E) embryos obtained by crossing heterozygous flh^-/+ pair. Expression of both ehh and Ntl is expanded in the tbx-c+ injected wild-type sibling embryo (E) and absent in the tbx-c+ injected ntl^-/- mutant embryo (D). (F) tbx-c expression in the flh^-/- embryo. The notochord is absent and tbx-c is undetectable in the midline region of flh^-/-embryo. (G,H) ntl expression in the flh^-/- embryo (G) and the flh^-/- embryo injected with tbx-c+ (H). A few ntl positive cells (arrows) can be seen in the midline of both group of embryos. (I,J) tbx-c expression in wild-type (I) and spt^-/- embryos (J) at 14 hpf. Arrows indicate the tbx-c expression in the notochord. Insert (C,E): high magnification to show co-expression of ehh and Ntl in the notochord. Scale bars, 100 μm.

(A-G) Analysis of expression of ntl and tbx-c in animal cap explants. Expression of ntl (A,C) and tbx-c was not detected in the control caps dissected from the uninjected embryos. Injection of activin mRNA induces expression of ntl (B) and tbx-c (D). Injection of tbxc+ induces ntl expression (E) and injection of ntl induces tbx-c expression (F). ntl is induced in caps after coinjection of activin and dn-tbx-c mRNA (G). Scale bar, 100 μm.

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