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Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides.

Rana AA, Collart C, Gilchrist MJ, Smith JC - PLoS Genet. (2006)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
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.

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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.
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pgen-0020193-g002: 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.

Mentions: How specific are the phenotypes we observe? At the end of this paper we address this point experimentally for a group of MOs that causes defects in gastrulation, but some general comments are necessary before describing the results we obtain. First, it is unlikely that our MOs exert toxic effects, because injection of 30 ng of a standard control MO has little or no effect on development (see, for example, Figure 2A, 2D, and 2G), and of the 262 MOs injected in the course of this work (some of which are “second site” MOs and excluding the additional MOs, see below), 89 have no effect on development (other than occasionally causing a slight delay) even at the higher dose of 30 ng (Table 1). We also note that MOs that are altered by five bases from their target sequences have little or no effect on development.


Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides.

Rana AA, Collart C, Gilchrist MJ, Smith JC - PLoS Genet. (2006)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636699&req=5

pgen-0020193-g002: 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.
Mentions: How specific are the phenotypes we observe? At the end of this paper we address this point experimentally for a group of MOs that causes defects in gastrulation, but some general comments are necessary before describing the results we obtain. First, it is unlikely that our MOs exert toxic effects, because injection of 30 ng of a standard control MO has little or no effect on development (see, for example, Figure 2A, 2D, and 2G), and of the 262 MOs injected in the course of this work (some of which are “second site” MOs and excluding the additional MOs, see below), 89 have no effect on development (other than occasionally causing a slight delay) even at the higher dose of 30 ng (Table 1). We also note that MOs that are altered by five bases from their target sequences have little or no effect on development.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus