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First published online 13 May 2004
doi: 10.1242/dev.01157

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A role for MKP3 in axial patterning of the zebrafish embryo

¹ Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (77K): [in a new window]   Fig. 1. Predicted amino acid alignment of MKP3 from human, mouse, Xenopus, Takifugu, zebrafish, Drosophila and chick. Phosphatase and ERK binding domains are underlined by red and blue, respectively. Residues marked in blue asterisks indicate the absolutely conserved amino acids found in all vertebrate MKPs within the ERK-binding domain.

View larger version (98K): [in a new window]   Fig. 2. Embryonic expression of mkp3 in zebrafish. (A-P) Lateral views, except in D and Q-T, animal view. (A) mkp3 is not expressed as a maternal transcript, but is initiated at the high stage (B). (C,D) At 30% epiboly, expression marks the margin. (E-H) During somitogenesis stages, mkp3 is restricted to domains where FGF genes are expressed, such as anterodorsal head, the MHB, rhombomere 4 and the caudal region. (I) Comparison of mkp3 (orange) with the ventral marker, vega2/vent (blue). (J-P) mkp3 expression is regulated by FGF signaling. (J-L) Sphere stage. Inhibition of FGF activity via ectopic expression of XFD, a dominant-negative FGF receptor, suppresses mkp3 expression (K), while hyperactivation of the FGF pathway through the expression of fgf8 induces an expansion in mkp3 expression (L). (M-P) 24 hpf. Expression of mkp3 is lost in the MHB (arrows) in ace, a fgf8 mutant (N), and in noi, a pax2.1 mutant (P). (Q-T) mkp3 expression is regulated by the RAS/MAPK pathway. The mkp3 3'UTR was used to detect endogenous mkp3 expression at 30% epiboly in uninjected (Q), DNRas-(R), mkp3-(S) and DNAkt (T)-injected embryos. mkp3 expression was suppressed in DNRas and mkp3-injected embryos, but was unaffected in DNAkt-injected embryos.

View larger version (74K): [in a new window]   Fig. 3. Initiation of mkp3 expression by maternal ß-catenin. (A) mkp3 is not expressed maternally but is initiated at the high stage at 3.3 hpf, as determined by RT-PCR; ß-actin and Histone H4 serve as loading controls. (B) Activity of the MAPK pathway in the early zebrafish embryo as visualized by staining with anti-phospho MAPK, is detected from the oblong stage onwards. (C,D) Both fgf3 and fgf8 are first expressed in the prospective organizer region at the oblong stage. (E,F) Immunohistochemical staining of MAPK-p shows it to be localized to the prospective organizer side of the oblong stage embryo (arrow in F), but not at 512 cell stage (E). (G-J) Expression of XFD does not inhibit the initiation of mkp3 expression at the high stage (G,H), but is effective at the dome stage (I,J). (K-N) High stage. mkp3 expression is expanded by activating the ß-catenin pathway through LiCl treatment (compare L with K), and by ectopic expression of an activated form of ß-catenin (N), whereas expression of a dominant-negative version of TCF3 suppresses the induction of mkp3 (M). (O-R) Sphere stage. The initial expression of mkp3 is abolished in the ich mutant (compare O with P). (Q) Activation of mkp3 by FGF genes is independent of ß-catenin, as ectopic expression of fgf8 in ich mutants can still activate mkp3. (R) Expression of mkp3 is unaffected in boz mutants, an organizer-defective mutant downstream of the maternal ß-catenin pathway. Arrowhead in N indicates ectopic mkp3 expression.

View larger version (79K): [in a new window]   Fig. 4. Ectopic expression of MKP3 blocks RAS/MAPK signaling. (A) mkp3 expression constructs. Sequence represents the phosphatase active site. (B) Xenopus animal explant assay. Explants injected with activated forms of RAS exhibit induction of Xbra, while co-injection of mkp3 suppresses RAS activity. The inactive mutant construct mkp3::C292S had no effect on the activated form of RAS. (C-J) Ectopic expression of mkp3 or mkp3::C292S can suppress or enhance, respectively, the expression of sef and spry4 at gastrula stage.

View larger version (101K): [in a new window]   Fig. 5. Phenotypes of zebrafish embryos injected with mkp3 and mkp3::C292S RNA. (A-C) Somitogenesis stages of zebrafish embryos uninjected (A), injected with mkp3 (B) or with mkp3::C292S (C). Injected embryos display anterior defects (red arrowhead). (D-I) At 24 hpf, mkp3-injected embryos (E) exhibit anterior truncation and expanded trunk-tail region, while mkp3::C292S-injected embryos, show trunk-tail defects (F) (compare with uninjected in D). (G-I) Staining for gata1 at 24 hpf reveals ventralization of mkp3-injected (H) and dorsalization in mkp3::C292S-injected embryos (I); arrows indicate region of gata1 staining (compare with G, uninjected). (J-Q) Early dorsoventral markers are disrupted in injected embryos. bmp4 is activated in mkp3-injected embryos (J,K, 70% epiboly), while chordin expression is suppressed (L,M, shield stage). Conversely, ectopic expression of mkp3::C292S expands expression of chordin at shield stage (N,O; arrowheads indicate the boundary of chd expression), and expression of neural markers en3 and krox20 (P,Q) at one-somite stage. MHB, mid/hindbrain boundary; r3 and r5, rhombomeres 3 and 5.

View larger version (74K): [in a new window]   Fig. 6. MKP3 is required in the early embryo for establishment of axial polarity. (A,D,F,H,J,L) Control MO (contMO) or 5 bp mismatch mkp3 MO (misMO)-injected embryos. (B,C,E,G,I,K,M) mkp3MO-injected embryos. Injection of mkp3MO results in dorsalized embryos (compare B and E with A and D). (C) The mkp3MO effects can be rescued by co-injection of HA::mkp3 that is insensitive to mkp3MO. (F-M) mkp3MO disrupts early dorsoventral patterning of the embryo, as shown by suppression of bmp4 (compare F with G), and expansion of chordin and fgf8 (arrows) (compare I with H and K with J). Neural markers en3 and krox20 are expanded in mkp3MO-injected embryos (compare L with M).

View larger version (62K): [in a new window]   Fig. 7. Expression of FGF genes in ich mutant embryos and rescue of phenotype by FGF. (A-M) Mutant embryos derived from ich-/- females and wild-type embryos derived from ich+/- females are shown as animal pole views. Initial expression of fgf3 and fgf8 is strongly reduced at sphere stage in mutant embryos (B,D) compared with wild-type embryos (A,C), where the double arrows mark dorsal expression. (E-M) Expression of fgf3 continues to be reduced in mutant embryos at 30% (E,F) and 50% epiboly (I,J). By contrast, fgf8 expression is detected at 30% epiboly around the margin (G,H), and continues in mutant and wild-type embryos at 50% (K,M). (N-O) Overexpression of fgf3 (compare N with O) or fgf8 (compare N with P) partially rescues ich embryos (asterisks indicates rescued embryos). (Q) Rescue of ich by injection of FGF genes and ß-catenin. Both fgf3 and fgf8 injections resulted in partial rescue of mutant embryos. RNA encoding the nuclear localized form of ß-galactosidase (nß-gal) or zebrafish ß-catenin was injected into ich embryos as a negative and positive control, respectively. Phenotypic classification was slightly modified from Kelly et al. (Kelly et al., 2000): class 1 embryos are the most ventralized, lacking trunk and head; class 1a develop partial trunk and spinal cord but no hindbrain or more anterior structures; class 2 develop spinal cord and hindbrain but not midbrain, forebrain or eyes; class 3 develop incomplete anterior brain and eye structures; class 4 develop a complete AP axis but no notochord; class 5 appear normal at 24 hpf.