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Vertebrate hoxc8 homologous genes have been shown to be involved in the formation of lower thoracic/lumbar vertebrae during early embryonic development. We report the isolation of a Xenopus hoxc8 (Xhoxc8), which shows 94% amino acid sequence identity to the mouse counterpart. Xhoxc8 is initially expressed in a broad region of blastopore lip at gastrular stage; however, at later stages, the region of expression is progressively restricted to the dorsal region caudal to the third somite and to the central trunk region of abdomen. Retinoic acid treatment that caused a severe malformation in antero-posterior axis did not induce any significant change in the spatio-temporal expression pattern of Xhoxc8 mRNA. Antisense RNA injection into 2- or 4-cell stage embryos resulted in a severe malformation in the abdominal structure leading to embryonic death. The results strongly indicate that Xhoxc8 expression is critical for the formation of abdominal structure.
Fig. 1.
Nucleotide and deduced amino acid sequences of Xhoxc8 mRNA. (A) Deduced amino acid sequences are indicated. EcoRI (AAGCTT) and HindIII (GAATTC) sites are underlined. These sequences are deposited in the GenBank database under Accession No. AF001596. (B) Protein sequence comparison among different species. Sequences of mouse [18] and human [19] were compared with Xenopus sequence. Dash lines indicate the identical amino acids; bold letters indicate homeobox; hexapeptide region is lightly shaded; polyglutamic acid stretch is heavily shaded.
Fig. 2.
Northern (A) and Southern blot (B) analyses for the determination of Xhoxc8 mRNA size and Xhoxc8 gene-copy number. For Northern blot, 20 μg of stage 20 total RNA was loaded on to the 1.2% agarose gel, which was run and transferred to nylon membrane in 10à SSC. The blot was hybridized with DIG-labeled RNA probe prepared from linealized pBXhoxc8w DNA template. For Southern blot, 10 μg of restriction enzyme digests of genomic DNA was size-fractionated on 1% agarose gel in TAE buffer and transferred to nylon membrane. Blots were hybridized with the same probe used in Northern blot analysis.
Fig. 3.
Expression pattern of Xhoxc8 analyzed by RT-PCR. (A) One μg of stage 0â35 total RNA was reverse transcribed and PCR amplified using Xhoxc8 specific primer. (B) Expression of Xhoxc8 along A-P at stage 30 and comparison of expressions with region-specific marker genes. Total RNAs for analyses were extracted from dissected parts of stage 30 embryos (Head, M1, M2, and M3) or from a whole embryo (Whole). Ef1-α and α-cardiac actin were used for internal control. (C) Vertical lines indicate the dissected regions.
Fig. 4.
In vivo Xhoxc8 expression pattern. Whole-mount in situ hybridization was performed using the same probe used for Northern and Southern blot analyses. (A) Ventral view of stage 10 embryo. Dorsal lip is indicated by arrow. (B) Dorsal view of stage 12 embryo. YP, yolk plug. (C) Lateral view of stage 25 embryo. (D) Lateral view of stage 30 embryo. (E) Lateral view of stage 35 embryo. (F) Lateral view of stage 37 embryo. Open arrowheads indicate the anterior limits of Xhoxc8 mRNA expression. White arrows indicate the anterior and posterior margins of the migrating neural crest cells. (G) Transverse section of stage 30 embryo following in situ hybridization. Notable tissues were indicated.
Fig. 5.
Effects of retinoic acid treatment on the Xhoxc8 expression. Embryos were treated with all-trans retinoic acid (RA, 1 or 10 μM) or dimethyl sulfoxide (DMSO). Stage 5 embryos were cultured in 0.1à MMR containing DMSO or RA until they reached stage 12. At stage 12, embryos were washed several times with 0.1à MMR and then cultured in the media until they reached stage 25 or 33. The embryos were fixed in MEMFA and the Xhoxc8 expression level was examined by whole-mount in situ hybridization.
Fig. 6.
Effects of Xhoxc8 antisense RNA injection. Capped synthetic sense and antisense Xhoxc8 RNAs were produced from linealized pBXhoxcw8 plasmid. Total 1 ng of sense (AâD) or antisense Xhoxc8 (EâH) RNA was injected into each blastomere of 2- or 4-cell stage embryos. The RNA injected animals were analyzed at st. 12 (A,E), 35 (B,F), 41 (C,G), and 48 (D,H).
