|
Fig. 1. Temporal and spatial expression of Xlphn2. (A) RT-PCR analysis was performed at various developmental stages. Lanes indicate stages according to Nieuwkoop and Faber (1956), except for F, which represents fertilized embryo. (B-E) Whole mount in situ hybridization reveals the spatial expression of Xlphn2 (B,C) Lateral view of stage 23 (B) or stage 30 (C) embryos stained with antisense probe of Xlphn2. (D,E) Lateral view of stage 23 (D) or stage 30 (E) embryos stained with sense probe of Xlphn2. Pla, preplacode; Pr, pronephros; e, eyes; MB, midbrain; R, rhombomeres; 1-4, epibranchial placode 1-4.
|
|
Fig. 2 (left). Phenotypes of Xlphn2 MO-injected embryos. (A-F) Morphology of morpholino antisense oligonucleotide (MO)-injected embryos at stage 35 (A-D) and stage 40 (E,F). Forty nanograms of control MO (A,E), 40 ng of Xlphn2 MO (B,F), or 40 ng of Xlphn2 MO with 1 ng of human LPHN2-FLAG mRNA (C) was injected into the animal pole of 2-cell stage embryos. The embryos were grown until desired stages to assess morphology. Uninjected sibling control is shown in (D). (A) n=52, normal morphology 100%; (B) n=57, reduced head structure with bent axis formation 95%; (C) n=20, normal morphology 85%; (D) n=84, normal morphology 100%. (E,F) N-CAM staining of control MO (E), or Xlphn2 MO (F)-injected stage 35 embryos. Black arrowheads show staining of the eye region. (G,H) Morphology of control MO (G) and Xlphn2 MO (H)-injected embryos at stage 40 (n=23 each). (I,J). Alcian blue staining of control MO (I), or Xlphn2 MO (J)-injected stage 45 embryos. M, meckel’s cartilage; C, ceratohyal cartilage; B, branchial cartilage.
|
|
Fig. 3 (right). Whole mount in situ hybridization of neural crest genes. Forthy ng of control MO (A,C,E,G) or Xlphn2 MO (B,D,F,H) was injected into the animal pole of 2-cell stage embryos. The embryos were grown until they reached the desired stage for WISH. Xtwist (A-D) and Xdlx-2 (E-H) were used as probes for staining stage 23 (A, B, E, F) and 30 (C, D, G, H) embryos. Streams of neural crest cells in the branchial arches are indicated by red arrowheads.
|
|
Fig. 4 (left). Whole mount in situ hybridization of the placode marker. Forty ng of control MO (A,C,E,G) or Xlphn2 MO (B,D,F,H) was injected into the animal pole of 2-cell stage embryos. The embryos were grown until they reached desired stages for WISH. Stage 23 and 30 embryos were stained with Xfoxi4.1 as a probe (A-D). (A,B) Stage 23, (C,D) stage 30, and (E,F) stage 30 embryos double-stained with Xtwist (light blue) and Xfoxi4.1 (purple). (G,H). A magnified view of a double-stained embryo indicated as box in (E,F) was shown. Black arrowheads, presumtive placode region; e, eyes; 1–4, epibranchial placode 1–4; yellow arrowhead, mandibular arch neural crest cells; green arrowhead, hyoid arch neural crest cells; red arrowhead, branchial arch neural crest cells
|
|
Fig. 5 (right). Neural crest (NC) cells were transplanted into stage 20 embryos, and photographed at stage 30 with fluorescent imaging. (A-F) tdTom-injected NC cells were transplanted into control (Ctl) MO-injected (A-C, n=22) or Xlphn2 (Xl2) MO-injected embryos (D-F, n=22). (B,E) tdTomato fluorescent images are shown. (C,F) Merged images are shown. Light blue arrowhead, unmigrated NC; yellow arrowhead, migrated NC. (G-L) Control (Ctl) MO+tdTom (G-I, n=11) or Xlphn2 (Xl2) MO+tdTom (J-L, n=12) -injected NC cells were transplanted into wild-type host embryos. (H, K) tdTomato fluorescent images are shown. (I,L) Merged images are shown. Light blue arrowhead, unmigrated NC; yellow arrowhead, migrated NC. (M) Statistical analysis of transplanted embryos was calculated. The percentages of migrated NCs in different experiments are shown as means with standard error.
The numbers of distinct streams containing tdTom-labeled NCs were counted and added to the bar graph. Statistical analyses against samples of the control MO-injected host transplanted with tdTom-injected NC cells were performed using Fisherâs exact t-test to determine statistical significance using the total number of transplanted embryos. **p< 0.01 was considered significant.
|
|
Fig. S1. (A-E) Genomic structure of Latrophilin-related genes in Xenopus laevis. Genomic region surrounding 500 kbp of Xenopus latrophilin-related genes is shown. Graphics were adapted and modified from X.laevis 9.1. on GB browser. Note that Xlphn3-like is not mapped completely on this region by JGI model because of low homology to human LPHN3.
|
|
Fig. S2. (A) Analysis of the protein sequence coded by Xlphn2. Xlphn2 (4215 base pair long) protein contains a galactose binding lectin domain (lectin), an olfactomedin-like domain (olf), a G-protein-coupled receptor proteolytic site (GPS), and a seven transmembrane receptor domain (7TM domain). (B)
Phylogenetic tree of Xlphn2 protein. Protein sequences of latrophilin-related gene products were analyzed by the Neighbor joining method. Percentages of identical amino acids (Identity) to the human LPHN2 protein are shown.
|
|
Fig. S3. (A-D) One hundred picograms of EGFP (A,B) or Xlphn2 5’UTR-EGFP (C, D: containing Xlphn2 MO sequence upstream to ATG codon of EGFP mRNA was co-injected with 20 ng of control MO (A,C) or Xlphn2 MO (B,D) into one blastomere of 2-cell stage embryos. The injected embryos were cultured until stage 20 for microscopical observation of fluorescence.
|
|
Fig. S4. qRT-PCR of stage 30 embryos. Forthy ng of control MO or Xlphn2 MO was injected into the animal pole of 2-cell stage embryos. The embryo was grown until stage 30 and total RNA was isolated from whole embryos to make cDNA for qRT-PCR. The expression level of each gene was measured using the relative quantification method with Elongation factor 1a (EF1a) as a reference. cDNA of pooled embryos (stage 30) was used as a standard, and the expression levels of these embryos was set to 1. At least five individual embryos were used for quantification and statistical analysis. **P<0.01 and *P<0.05 were considered significant
|
|
Fig. S5. Xlphn2 is not required for induction of neural crest cells. (A,B) Stage 15 embryos were stained with Xtwist as a probe. Lateral view is shown.
|
