XB-ART-34948PLoS Genet. November 17, 2006; 2 (11): e193.
Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides.
To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between -80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing "rescue" experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into "synphenotype groups," members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.
PubMed ID: 17112317
PMC ID: PMC1636699
Article link: PLoS Genet.
Grant support: Wellcome Trust
Genes referenced: acbd6 actr6 aftph ahnak ap1m1 ap1m2 ap2m1 ap4s1 ap5s1 arfgap2 arid4a arl8a aurka aurkaip1 aurkb baz1b bmp4 brwd3 c1orf131 calhm2 cbx1 ccdc174 cda cdc42 cdh3 cdk2ap2 cdx1 cdx2 celsr1 cep295 cers2 cers3 chn1 clint1 cpsf1 cpsf2 cpsf3 cpsf4 csrnp1 dctn2 dlg1 dmd.1 dmd.2 dnal1 dnal4 dnali1 dock7 dync1i2 dync1li1 dync2h1 dync2li1 dynll1 dynlt1 eaf2 eed elk4 eogt eomes ext1 fam214a fgfr1 fgfr3 fgfr4 fzd10 fzd2 fzd6 fzd7 fzd8 hap1 hbegf heatr5b hesx1 hmg20b hmgn2 hoxb3 hoxc8 hoxd1 ing2 ing3 ing5 inhbb itgb1 iws1 kdm2a kiaa1841 klf11 kmt5b kmt5c lefty lhx1 loc100135120 loc100144286 loc100489386 loc733728 loc780754 lpar1 lzts2 mark2 mau2 mfge8 mga miga2 mmadhc msx1 myo18a myo18b ncaph2 nipbl nodal1 nodal3.1 nog2 nradd nsa2 nsrp1 otog otx2 padi2 pard3b pard6b pard6g pced1a pdss2 pinx1 plekha1 prdm1 prkci r3hdm2 rac1 rad51 rb1 rbl2 rcbtb2 reep4 rhbdd3 rnf112 rpap3 rtf2 rxrb sass6 sccpdh serpina3 serpinb6 serpine2 sfrp1 skida1 smad1 smad2 smad3 smad4.1 smad4.2 smad6 smad7 smad9 smarca2 spata6l spice1 st13 sugt1 suv39h1 suv39h2 t2 tbx2 tbx3 tbx6 tbxt tcaf1 tceanc2 tgfbrap1 tgif1 tgif2 timm29 tmem70 tp53 tp53bp1 trmt61a tsg101 uba5 ubn2 ubtd2 ugt3a2 vegt vhl wdr74 wdtc1 wnt11 wnt11b wnt5b wnt8a ywhab ywhae ywhah ywhaq ywhaz zbed4 zmynd11 znf207 znf326 znf500 znf674 zup1
Morpholinos referenced: acbd6 MO1 acbd6 MO2 actr6 MO1 aftph MO1 ahnak MO1 ahnak MO2 ap1m1 MO1 ap1m2 MO1 ap2m1 MO2 ap4s1 MO1 ap5s1 MO1 ap5s1 MO2 arfgap2 MO1 arid4a MO1 arid4a MO2 arl8a MO(1 aurka MO1 aurkaip1 MO1 aurkb MO1 baz1b MO2 bmp4 MO1 brwd3 MO1 c19orf52 MO1 c19orf52 MO2 c1orf131 MO1 calhm2 MO1 calhm2 MO2 cbx1 MO1 ccdc174 MO1 ccdc174 MO2 cda MO1 cdc42 MO1 cdh3 MO2 cdk2ap2 MO1 cdx1 MO3 cdx2 MO2 cdx2 MO3 celsr1 MO1 celsr1 MO2 cep295 or kiaa1731 MO1 cep295 or kiaa1731 MO2 cers2 MO1 cers3 MO1 chn1 MO2 clint1 MO1 cpsf1 MO1 cpsf2 MO1 cpsf3 MO1 cpsf4 MO1 csrnp1 MO1 dctn2 MO1 dctn2 MO2 dlg1 MO1 dmd.1 MO1 dmd.1 MO2 dmd.2 MO1 dmd.2 MO2 dnal1 MO1 dnal1 MO2 dnal4 MO1 dnal4 MO2 dnali1 MO1 dock7 MO1 dync1i2 MO1 dync1li1 MO2 dync1li1 MO3 dync2h1 MO1 dync2h1 MO2 dync2li1 MO1 dync2li1 MO2 dynll1 MO1 dynlt1 MO1 dynlt1 MO2 eaf2 MO1 eed MO1 elk4 MO2 eogt MO1 eomes MO3 eomes MO4 ext1 MO2 fam214a MO1 fam214a MO2 fam73b fgfr1 MO4 fgfr3 MO1 fgfr3 MO2 fgfr4 MO5 fgfr4 MO6 fzd10 MO2 fzd10 MO3 fzd2 MO2 fzd2 MO3 fzd6 MO1 fzd6 MO2 fzd7 MO6 fzd7 MO7 fzd8 MO4 fzd8 MO5 hap1 MO1 hbegf MO1 heatr5b MO1 heatr5b MO2 hesx1 MO1 hmg20b MO1 hmgn2 MO2 hoxb3 MO1 hoxc8 MO1 hoxd1 MO3 ing2 MO1 ing3 MO1 ing5 MO1 inhbb MO1 inhbb MO2 itgb1 MO3 iws1 MO1 kdm2a MO2 kiaa1841 MO1 klf11 MO1 lefty MO8 lhx1 MO2 loc100135120 MO1 loc100144286 MO1 loc100489386 MO1 loc100489386 MO2 loc733728 MO1 lzts2 MO1 mark2 MO4 mau2 MO1 mfge8 MO1 mga MO1 mga MO2 mmadhc MO1 msx1 MO4 myo18a MO1 myo18b MO1 myo18b MO2 ncaph2 MO1 ncaph2 MO2 nipbl MO1 nipbl MO2 nodal1 MO7 nodal1 MO8 nodal3.1 MO2 nodal3.1 MO3 nog2 MO2 nradd MO1 nsa2 MO1 nsrp1 MO1 otog MO2 otog MO3 otx2 MO3 padi2 MO1 pard3b MO1 pard6b MO2 pard6g MO1 pced1a MO2 pdss2 MO1 pinx1 MO1 plekha1 MO1 prdm1 MO1 prkci MO2 r3hdm2 MO2 rac1 MO2 rad51 MO1 rb1 MO2 rbl2 MO1 rcbtb2 MO1 reep4 MO2 reep4 MO3 rhbdd3 MO1 rnf112 MO1 rpap3 MO1 rtfdc1 MO1 rtfdc1 MO2 rxrb MO1 sass6 MO1 sccpdh MO1 serpina3 MO1 serpinb6 MO1 serpine2 MO4 sfrp1 MO4 sfrp1 MO5 skida1 MO1 smad1 MO1 smad2 MO3 smad3 MO1 smad4.1 MO2 smad4.2 MO3 smad6 MO2 smad7 MO3 smad9 MO2 smarca2 MO2 spata6l MO1 spice1 MO1 spice1 MO2 st13 MO1 sugt1 MO1 sugt1 MO2 suv39h1 MO1 suv39h2 MO1 suv420h1 MO2 suv420h1 MO3 suv420h2 MO3 t MO3 t MO4 t2 MO3 tbx2 MO3 tbx2 MO4 tbx3 MO1 tbx3 MO2 tbx6 MO5 tbx6 MO6 tcaf1 MO1 tceanc2 MO1 tgfbrap1 MO1 tgif1 MO1 tgif2 MO3 tmem70 MO1 tmem70 MO2 tp53 MO4 tp53bp1 MO1 trmt61a tsg101 MO1 uba5 MO1 ubn2 MO1 ubn2 MO2 ubtd2 MO1 ugt3a2 vegt MO10 vegt MO2 vegt MO9 vhl MO1 wdr74 MO1 wdtc1 MO1 wnt11b MO5 wnt11b MO6 wnt5b MO1 wnt5b MO2 wnt8a MO5 wnt8a MO6 ywhab MO1 ywhab MO2 ywhae MO1 ywhae MO2 ywhah MO1 ywhah MO2 ywhaq MO1 ywhaq MO2 ywhaz MO1 zbed4 MO1 zmynd11 MO1 zmynd11 MO2 znf207 MO1 znf326 MO1 znf500 MO1 maybe znf674 MO1 znf674 MO2 zufsp MO1
Article Images: [+] show captions
|Figure 1. Embryos of X. tropicalis at the Stages Examined for Abnormalities Caused by Injection of MOs Embryos had been injected at the one-cell stage with a Lissamine-labeled control MO. (A�C) Bright field views. (D�F) Fluorescent views. D' shows a high-power view of cells within the animal hemisphere of an embryo at the early gastrula stage. (A and D) Early gastrula stage 10�11. (B and E) Tailbud stage 28. (C and F) Tadpole stage 41. doi:10.1371/journal.pgen.0020193.g001|
|Figure 2. Examples of the Similarities between Phenotypes Caused by Second Site MOs and Those of the Primary MO Directed against Sequence around the Translation Start Site of the Target mRNA (A�C) MOs directed against 14-3-3η. (A) Control MO; embryos develop normally. (B) Embryos injected with MO1, directed against the translation start site of 14-3-3η, develop with a shortened antero-posterior axis. (C) Embryos injected with MO2, directed against sequence 5′ of the translation start site of 14-3-3η, resemble those injected with MO1. (D�F) MOs directed against Xnr3. (D) Control MO; embryos develop normally. (E) Embryos injected with MO1, directed against the translation start site of Xnr3, exhibit an upturned tail. (F) Embryos injected with MO2, directed against sequence 5′ of the translation start site of Xnr3, also have an upturned tail, but they differ slightly from those injected with MO1 because their antero-posterior axes are slightly shortened. (G�J) MOs directed against Tbx3. (G and I) Embryos injected with control MOs develop normally. (H) Embryos injected with MO1, directed against the translation start site of Tbx3, have a normal body axis but their tails are slightly wavy. (J) Embryos injected with MO2, directed against sequence 5′ of the translation start site of Tbx3, have a more severe phenotype than those injected with MO1, in which the antero-posterior axis of the embryo is shortened. MO1, primary MOs; MO2, second site MOs. doi:10.1371/journal.pgen.0020193.g002|
|Figure 3. The Phenotypic Classes and Synphenotype Groups Defined by This Work doi:10.1371/journal.pgen.0020193.g003|
|Figure 4. The Nine Members of the Gastrula Defect Phenotypic Class Note that the blastopore in control embryos is closing normally, but is either absent or severely delayed in embryos in which the functions of the indicated genes are inhibited. All embryos shown are at gastrula stage 10.5 to 11.5. In this figure and in Figures 5�13, the number in the top left hand side of each panel represents the synphenotype group to which the embryos belong, and the name of the gene in question is shown bottom left. doi:10.1371/journal.pgen.0020193.g004|
|Figure 5. The First 32 Members of the Shortened Axis Phenotypic Class This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. Members of the first three synphenotype groups (involution defects, gastrula or neurula defects, and short axis surviving to tailbud) are shown at tailbud stage (stage 24�28), while the member of the second synphenotype group shown here (short axis surviving to tadpole) is shown at tadpole stage 35�41. Lines in this and subsequent figures demarcate the different synexpression groups. doi:10.1371/journal.pgen.0020193.g005|
|Figure 6. The Second 32 Members of the Shortened Axis Phenotypic Class This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. The figure shows examples of the fourth synphenotype group (short axis surviving to tadpole) at tadpole stages 35�41. doi:10.1371/journal.pgen.0020193.g006|
|Figure 7. The Final 14 Members of the Shortened Axis Phenotypic Class This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. The figure shows examples of the second three synphenotype groups (short axis surviving to tadpole, normal body short tail, and proportionately small) at tadpole stages 35�41. doi:10.1371/journal.pgen.0020193.g007|
|Figure 8. CPSF4, the Sole Member of the Late Degradation Phenotypic Class Embryos injected with MOs targeting this gene appear perfectly normal to early tailbud stage 30 but then rapidly disintegrate. Embryos are shown at the tailbud stage (stage 24�28). doi:10.1371/journal.pgen.0020193.g008|
|Figure 9. The Three Members of the Curved Body Axis Phenotypic Class Embryos are shown at the tadpole stage (stage 35�41). doi:10.1371/journal.pgen.0020193.g009 Ventral tissue defects.|
|Figure 10. The Five Members of the Ventral Defects Phenotypic Class This class can be subdivided into two synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35�41). doi:10.1371/journal.pgen.0020193.g010|
|Figure 11. The First 32 Members of the Bent Axis Phenotypic Class This class can be subdivided into five synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35�41). doi:10.1371/journal.pgen.0020193.g011|
|Figure 12. The Final 31 Members of the Bent Axis Phenotypic Class This class can be subdivided into five synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35�41). doi:10.1371/journal.pgen.0020193.g012|
|Figure 13. The 14 Members of the Motility Defects Phenotypic Class This class can be subdivided into three synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35�41). doi:10.1371/journal.pgen.0020193.g013|
|Figure 14. Apoptosis Is Not a Non-Specific Response to Injection of MOs Embryos were injected at the one-cell stage with 15 ng of the indicated MO and allowed to develop to the equivalent of the tailbud stage, when they were examined by TUNEL staining. Note that only the Tbx3.2, Xnr3.2, and Xbra.2 MOs caused a level of apoptosis that exceeded the level observed in control embryos (injected with 30 ng of the Gene Tools control MO). doi:10.1371/journal.pgen.0020193.g014|
|Figure 15. Tests of the Specificities of the Phenotypes Observed in the Gastrula Defects Phenotypic Class The specificities of the MOs used to define this phenotypic class were investigated by injecting 10�15 ng of the Gene Tools standard control MO (Column 1); the original antisense MO (Column 2); MO1 together with 1 ng of a form of the target RNA that lacks the MO target sequence (Column 3); MO1 (or, in the case of Dp71, MO2) with five mismatched bases (Column 4); MO2 (Column 5); MO2 together with 1 ng of a form of the target RNA that lacks the MO target sequence (Column 6). The results of these experiments are summarized in Table 5. In Column 1 (control MO) embryos are shown at the mid-gastrula stage. Embryos in Column 2 (MO1) are at the same stage as those in Column 1, but (with the exception of Dp71) gastrulation is delayed or inhibited. In the case of Dp71, MO1 does not inhibit gastrulation but does cause embryos to develop with a shortened axis. Column 3 indicates that for five of the nine MOs studied, complete or partial rescue of the phenotype was obtained by injection of the cognate RNA. In these experiments, embryos were allowed to develop beyond gastrula stages to tailbud or tadpole stages. In the case of D1LIC, rescue was more complete at tailbud stages (upper panel) than tadpole stages (lower panel). Column 4 shows that for each of the nine MOs, changing five bases caused them to lose the ability to disrupt development. Use of a second site MO usually causes a milder phenotype than is observed with MO1 (Column 5), but the phenotype is usually specific, in the sense that it can frequently be rescued by injection of the cognate RNA (Column 6). MO1, original antisense oligonucleotide; MO2, second site MO. doi:10.1371/journal.pgen.0020193.g015|
|Figure 16. Members of the Same Synphenotype Group Do Not Necessarily Have the Same Expression Patterns The six examples shown here are all from the motility defects class. (A�C) Expression patterns of the three members of the swimming in circles synphenotype group. (D�F) Expression patterns of three members of the normal appearance but paralyzed synphenotype group. Members of each group are not expressed in the same patterns and so do not belong to the same synexpression group (see text). doi:10.1371/journal.pgen.0020193.g016|
|ap1m (adaptor-related protein complex 1, mu 1 subunit) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.|
|uba5 (ubiquitin-like modifier activating enzyme 5 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.|