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The diploid frog X. tropicalis has recently been adopted as a model genetic system, but loss-of-function screens in Xenopus have not yet been performed. We have undertaken a pilot functional knockdown screen in X. tropicalis for genes involved in nervous system development by injecting antisense morpholino (MO) oligos directed against X. tropicalis mRNAs. Twenty-six genes with primary expression in the nervous system were selected as targets based on an expression screen previously conducted in X. laevis. Reproducible phenotypes were observed for six and for four of these, a second MO gave a similar result. One of these genes encodes a novel protein with previously unknown function. Knocking down this gene, designated pinhead, results in severe microcephaly, whereas, overexpression results in macrocephaly. Together with the early embryonic expression in the anterior neural plate, these data indicate that pinhead is a novel gene involved in controlling head development.
Figure 6. Overexpression of 25H12.1 (pinhead) causes enlarged heads. Xenopus laevis embryos were injected in each blastomere after the first cleavage with 1 ng of 25H12.1 sense mRNA containing the coding section of the gene together with 100 ng/blastomere of nuclear beta -gal mRNA as an injection tracer (blue stain). Pinhead-injected embryos show enhanced anterior development (d-f) compared with embryos injected with nuclear beta -gal alone (a-c), as well as some epidermal blistering in the trunk.
Figure 2. A: Reproducible phenotypes induced by morpholino (MO) injection. Control uninjected embryos and embryos injected with each MO, as indicated. All are hybridised with a neural marker (blue staining), apart from (p–s) which are unstained, as follows: a–d, ElrC; e,f, Sox3; g–o, N-tubulin. The main features of the phenotypes caused by the MOs were as follows: 16G8 causes shortening of the anterior–posterior axis, 26A10.2 causes a reduction of ElrC staining particularly in the cranial nerve region (arrowhead in d) and broadening of the hindbrain (arrowhead in f), 17D4 causes posterior truncation (h) and/or bifurcated axis (i), 26E1.1 results in enhanced posterior development/open proctodeum (arrowhead in k and l) and hypodorsalisation, 18E6 causes cell death that starts anteriorly (n) and spreads posteriorly (o), and 25H12.1 causes extreme microcephaly. Most are lateral views apart from e,f,m,n,o,r,s, which are dorsal views. Anterior is to the left in all panels. Each set of control and experimental embryos was photographed under the same magnification (×40–×60). B: Expression patterns of targeted genes by in situ hybridisation. a: Expression of 16G8 (blue) along the neural tube of a neurulaembryo. b: Expression of 16G8 (magenta) in a broad band around the equator of a gastrulating embryo, blastopore side down. c: 26A10.2 expression is high in neural tissue of neurula embryos. d: 17D4 expression in a neurulaembryo. e: 26E1.1 expressed at the anterior neural tube. f: 18E6 expression at the anterior end of the neural tube. g: 25H12.1 expression in mid-anterior neural plate in two bilateral patches (black arrowhead) and the anterior neural ridge (white arrowhead). h,i: 25H12.1 is expressed around the blastopore of gastrulating embryos but excludes the dorsal mesoderm highlighted by in situ hybridisation to chordin (in light blue). All neurula embryos are viewed from the dorsal side with anterior to the front.
Figure 4. Morpholinos (MOs) directed against 25H12.1 (pinhead) splice sites cause aberrant splicing and an increase in detectable pinhead mRNA. A: Diagram of part of the 25H12.1 gene in X. tropicalis with exonic sequences represented as boxes and intronic sections as black lines. I: The target sites of 25H12.1moA and 25H12.1moB at the boundary of a 179-bp intron and a 96-bp exon are shown as moA and moB. Primers f2 and r1 used for reverse transcriptase-polymerase chain reaction (RT-PCR) are depicted as arrows. II: Wild-type cDNA yields a 187-bp RT-PCR product. III,IV: Aberrantly spliced cDNAs yield 366-bp and 91-bp RT-PCR products with f2 and r1 due to inclusion of the 179-bp intron or skipping of the 96-bp exon. The gel shows RT-PCR (primers f2 and r1) of 25H12.1 cDNA from uninjected (WT, wild-type) neurula embryos or embryos injected at one-cell stage with 10 ng of 25H12.moA (moA) or 25H12.1moB (moB). Amplified DNA fragments are labelled (I–IV) according to the diagram shown above. Both MOs induce abnormal splicing. M, marker lane; C, no cDNA control; G, Xenopus tropicalis genomic DNA; P, plasmid DNA from TGas046h20 that includes insertion of the 179-bp intron. B:X. tropicalis embryos uninjected (a) or injected at the one-cell stage with 10 ng of 25H12.1moB (b,c) and hybridised with an 25H12.1 antisense RNA probe. As well as the pinhead phenotype, injected embryos show increased amounts of 25H12.1 mRNA in anterior regions (arrowheads in b) and around the tail bud (arrowhead in c).
