Takahashi C et al. (2012), Identification and characterization of Xenopus ...

XB-ART-45656

Int J Dev Biol 2012 Jan 01;565:393-402. doi: 10.1387/ijdb.113333ct.

Show Gene links Show Anatomy links

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway.

Takahashi C , Suzuki T , Nishida E , Kusakabe M .

???displayArticle.abstract???
The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.

???displayArticle.pubmedLink??? 22811273
???displayArticle.link??? Int J Dev Biol

Species referenced: Xenopus laevis
Genes referenced: cdx2 cdx4 fgfr1 foxd3 kctd15 msgn1 myc ncam1 odc1 pax3 sox10 sox2 tbx2 tbxt
???displayArticle.morpholinos??? kctd15 MO1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Evolutionary conservation of Xenopus kctd15, a novel FGF-repressed gene. (A) Xenopus Kctd15 expression is repressed by FGF or RasV12 in ectodermal explants. hRasV12 mRNA was injected into the animal poles of four-cell stage embryos. Ectodermal explants derived from uninjected or injected embryos were left untreated or treated with 100 ng/ml FGF. At stage 11, explants were harvested for RNA preparation. The expression level of kctd15 was determined by real-time quantitative RT-PCR and normalized relative to that of ef1a. Shown is the average of two independent experiments. The error bar represents the standard deviation (SD). Significant differences are marked by asterisks (p < 0.05, *p < 0.01, unpaired t test). P values versus control were as follows: 0.067 for FGF-treated explants, 0.0030 for hRasV12 mRNA (4 pg)-injected explants, and 0.016 for hRasV12 mRNA (10 pg)-injected explants. (B) Schematic representation of Xenopus kctd15 protein. Xenopus kctd15 consists of 255 amino acids and has the potassium channel tetramerization domain at the N-terminus. (C) A phylogenetic tree of vertebrate KCTD15 proteins. The tree was drawn by the BLAST TreeView Widget (http://blast.ncbi.nlm.nih.gov/). Genetic distances were calculated from the aligned sequences by using the Grishin distance model. The tree was built with the Fast Minimum Evolution method. Identity of amino acid sequences among vertebrate KCTD15 orthologs is shown in the table. (D) Alignment of vertebrate KCTD15 protein sequences. Asterisks mark amino acid residues conserved in all species. Periods mark amino acid residues conserved in at least 4 out of 7 species. (E) Temporal expression of Xenopus kctd15 in early development. The total RNA isolated from embryos at indicated stages was subjected to real-time quantitative RT-PCR. The expression level of kctd15 was normalized relative to that of odc. Shown is the average of two independent experiments. The error bar represents the standard deviation (SD).
	Fig. 2 Spatial expression of Xenopus kctd15 in early development. Whole mount in situ hybridization against kctd15 was performed on embryos from indicated stages. No detectable signal was seen with the sense probe (data not shown). (A-C) At stage 10.5, kctd15 was uniformly expressed throughout the presumptive ectoderm. (D-G) At stage 14, kctd15 expression was detected in the non-neural ectoderm and was excluded from the neural plate. Dorsal is upward in D and E. Anterior is upward in F and G. (H-J) At stage 16, kctd15 expression was detected in the preplacodal ectoderm (ppe), the neural crest (nc) and the roof plate (rf). Dorsal is upward in H and I. Anterior is upward in J. (K-M) At stage 18, kctd15 expression was detected in the neural crest (nc), the roof plate (rf) and the olfactory placode (pOl). Dorsal is upward in K and L. Anterior is upward in M. (N-P) At stage 25, kctd15 expression was detected in the pronephric duct (pd), the mandibular arch (Md), the hyoid arch (Hy), the olfactory placode (pOl), trigeminal ganglia (tg) and the anterodorsal lateral line placode (pAD). Anterior is left. (Q) Whole mount double in situ hybridization against kctd15 and sox10 was performed on stage 17 embryos (middle and right panels). The right panel is an enlarged view of the middle panel. Dark blue and orange dotted lines mark the kctd15-expressing and sox10-expressing area, respectively. For reference, single in situ hybridization against sox10 (red purple) was also performed (the left panel). All panels are dorsal views with anterior to the top.
	Fig. 3. FGF signaling inhibits Xenopus kctd15 expression in vivo. (A) FGF represses kctd15 expression indirectly. Animal caps were dissected at stage 10.25, treated with FGF (100 ng/ml) for 3 hours in the presence or absence of cycloheximide (CHX; 5 mg/ml), and then harvested for real-time quantitative RT-PCR analysis. Shown is the average of two independent experiments. The error bar represents SD. Significant differences are marked by asterisks (p < 0.05, unpaired t test). P values (FGF treatment versus no FGF treatment) were as follows: 0.024 for kctd15 expression in the absence of CHX, 0.80 for kctd15 expression in the presence of CHX, 0.23 for xbra expression in the absence of CHX, and 0.24 for xbra expression in the presence of CHX. (B) Inhibition of FGF signaling increases the abundance of kctd15 mRNA. XFD mRNA (1 ng) was injected into animal poles at 2-cell stage, and injected embryos were harvested at stage 11 for real-time quantitative RT-PCR analysis (left). Embryos were treated with indicated doses of SU5402 at stage 9, and then harvested at stage 11 for real-time quantitative RT-PCR analysis (right). Shown is the average of two (left) or three (right) independent experiments. The error bar represents SD. P < 0.05, unpaired t test. P values versus control were as follows: 0.14 for XFD mRNA-injected embryos, 0.031 for SU5402 (50 mM)-treated embryos, and 0.015 for SU5402 (100 mM)-treated embryos. (C) Inhibition of FGF signaling dramatically expands the region expressing kctd15. Embryos were treated with indicated doses of SU5402 at stage 9, fixed at stage 15, and then subjected for whole mount in situ hybridization against kctd15. Upper panels show dorsal views with anterior toward the top. Lower panels show lateral views with anterior to the left. White arrowheads indicate unclosed blastopores, which are commonly observed phenotypes caused by inhibition of the FGF pathway (Amaya et al., 1991; Chung et al., 2004).
	Fig. 4. Essential role of Xenopus kctd15 in dorsal development. (A) The specificity and efficiency of kctd15 MO. The combination of GFP mRNA (200 pg) plus control MO (80 ng) or kctd15 MO (80 ng) was co-injected with myc-kctd15 (800 pg) or kctd15-myc mRNA (800 pg) into Xenopus embryos. Injected embryos were harvested for immunoblotting at stage 10.5. (B,C) Control MO (80 ng) or kctd15 MO (80 ng) was injected into the animal pole of each dorsal blastomere at the four-cell stage. For rescue experiments, myc-kctd15 mRNA (800 pg) was coinjected with kctd15 MO (80 ng). At the late tailbud stage, injected embryos were photographed with anterior to the left and dorsal to the top. White dotted lines in C represent somite boundaries. (D) Obtained phenotypes were classified into three groups (severe, mild or normal) according to the extent of defects in head morphologies. , P < 0.01. Fisher exact test was used to compare the frequency of normal head development between embryos injected with kctd15 MO alone and those injected with kctd15 MO plus myc-kctd15 mRNA. P = 6.5 x 10-8. (E) Obtained phenotypes were classified into three groups (severe, mild or normal) according to the extent of defects in somite morphologies. , P < 0.01. Fisher exact test was used to compare the frequency of normal somite development between embryos injected with kctd15 MO alone and those injected with kctd15 MO plus myc-kctd15 mRNA. P = 0.00045. (F-I) Real-time quantitative RTCR analysis of marker gene expression. Embryos dorsally injected with control MO (80 ng) or kctd15 MO (80 ng) were cultured until stage 15 (F, G) or 19 (H, I). The relative expression levels of the indicated genes were normalized to that of odc. Shown is the average of two (F-H) or four (I) independent experiments. The error bar represents SD. P < 0.05, *P < 0.01, unpaired t test. P values versus control were as follows: 0.11 for foxd3 expression, 0.27 for sox10 expression, 0.0046 for pax3 expression, 0.31 for sox2 expression, 0.029 for ncam expression, 0.00083 for xbra expression, and 0.47 for mespo expression.
	Fig. 5. Essential role of Xenopus kctd15 in ventral development. (A) Control MO (80 ng) or kctd15 MO (80 ng) was injected into the animal pole of each ventral blastomere at the four-cell stage. For rescue experiments, myc-kctd15 mRNA (700 pg) was coinjected with kctd15 MO (80 ng). At the late tailbud stage, injected embryos were photographed with anterior to the left and dorsal to the top. (B) Obtained phenotypes were classified into three groups (severe, mild or normal) according to the extent of defects in ventral and caudal morphologies. *, P < 0.01. Fisher exact test was used to compare the frequency of normal development between embryos injected with kctd15 MO alone and those injected with kctd15 MO plus myc-kctd15 mRNA. P = 2.7 x 10-5. (C-E) Real-time quantitative RT-PCR analysis of marker gene expression. Embryos ventrally injected with control MO (80 ng) or kctd15 MO (80 ng) were cultured until stage 15 (C), 19 (D) or 24 (E). The relative expression levels of the indicated genes were normalized to that of odc. Shown is the average of two independent experiments. The error bar represents SD. P < 0.05, **P < 0.01, unpaired t test. P values versus control were as follows: 0.085 for foxd3 expression, 0.30 for sox10 expression, 0.00065 for pax3 expression, 0.0089 for xbra expression, and 0.48 for cdx2 expression, and 0.011 for cdx4 expression.
	Fig. 6. Ectopic dorsal expression of fgfr1 phenocopies dorsal depletion of Xenopus kctd15. (A) The indicated dose of fgfr1 WT mRNA was injected into the animal pole of each dorsal blastomere at the four-cell stage. Embryos were photographed at the late tailbud stage. (B) Obtained phenotypes were classified into three groups (severe, mild or normal) according to the extent of defects in head morphologies. (C) Obtained phenotypes were classified into four groups (no somite, severe, mild or normal) according to the extent of defects in somite formation. (D) The indicated dose of fgfr1 WT mRNA was injected into the animal pole of each ventral blastomere at the four-cell stage. Embryos were photographed at the late tailbud stage.
	kctd15 (potassium channel tetramerization domain containing 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, horizontal view, animal up.
	kctd15 (potassium channel tetramerization domain containing 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior left, dorsal up.
	kctd15 (potassium channel tetramerization domain containing 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.