XB-ART-15113Dev Growth Differ. February 1, 1998; 40 (1): 97-104.
Homeobox genes are expressed both temporally and spatially during vertebrate development, and regulate the tissue-specific expression of other genes. A Xenopus paired-related homeobox- 1 (Xprx-1) cDNA was cloned. Xprx-1 had a paired-related homeodomain, but did not contain a paired-box. The sequence of Xprx-1 had a high level of homology with K-2(mouse) and Prx-1 (chicken), thus Xprx-1 is assumed to be the Xenopus homolog of these genes. Xprx-1 transcripts were maternally restricted, in Xenopus embryos, and a decrease in the late blastula stage was followed by an increase in zygotic transcripts after gastrulation. The transcripts were localized to the animal hemisphere of the late blastula and were concentrated in the branchial arches of the tail-bud stage embryo. In animal cap experiments, Activin A dose-dependently induced Xprx-1 gene expression. These results suggest that Xprx-1 plays a role in early Xenopus development similar to other species.
PubMed ID: 9563915
Article link: Dev Growth Differ.
Genes referenced: fgf2 inhba prrx1
Article Images: [+] show captions
|Fig. 1. Sequence of Xprx-1 and comparison with related homeodomain proteins. (a) Sequence of Xprx-1 eDNA and the corresponding amino acid sequence. The homeodomain is boxed. Primers used for cloning and detection of expression are indicated by arrows. (b) Phylogenetic tree of Xprx-1 and related homeodomain proteins.|
|Fig. 2. Embryonic expression of Xprx-1. (a) Northern blot analysis was performed with total RNA (20 j..Jg) from embryos at stage 40. The full length Xprx-1 coding region was used as a probe. (b) RT-PCR and Southern blot analysis showing the temporal expression of Xprx-1. Total RNA for RT-PCR was isolated from five embryos at various stages. -7.48 Xprx-1• -4.40 -2.37 Xprx-1 ODC (egg) fertilized egg; (8) stage 8, mid-blastula; (9) stage 9, late blastula; (10) stage 10, early gastrula; (12) stage 12, late gastrula; (15) stage 15, mid-neurula; (20) stage 20, late neurula; (30) stage 30, tail-bud. Subcloned PCR products were sequenced and used as probes. The RT-PCR was performed under two conditions, 27 cycles (top) and 30 cycles (middle). ODC is a loading control that is uniformly expressed throughout early developmental stages (bottom; Bassez et a/. 1990; Osborne et at. 1991 ) . RT- (reverse transcriptase was not added to the total WE RNA) is a negative control, and no genomic DNA contamination was detected.|
|Fig. 3. Spatial expression of Xprx- 1 at stage 9. RT-PCR and Southern blot analysis showing localized expressions of Xprx-1. Fifty embryos were dissected into an animal cap (AC). vegetal cap (VC), dorsal marginal zone (DMZ), lateral marginal zone (LMZ). and ventral marginal zone (VMZ), as shown in the diagram at the right. Total RNA was isolated and used for RT-PCR and Southern blot analysis. EF1-a is an VC internal loading control that is expressed ubiquitously in embryos (Krieg et at. 1989). WE (whole embryo, stage 9) is a positive control, and RT- (reverse transcriptase was not added to the total WE RNA) is a negative control.|
|Fig. 4. Spatial expression of Xprx- 1 during tail-bud stages. Xprx-1 expression was detected by wholemount in situ hybridization using albino embryos. In (a)-( e), anterior is at the left. (a) Lateral view of stage 25 embryo. Xprx-1 is weakly expressed in the head region (arrowhead). (b) Lateral view of stage 30 embryo. Xprx-1 is expressed in head mesenchymal areas (arrowhead) and branchial arches (arrows, 1 ,2,3,4, are the arch numbers). (c) Enlarged view of (b). (d) Dorsal view of a stage 30 embryo. (e) Lateral view of a stage 35 embryo. Xprx-1 is expressed in the same region (left-arrowhead) and the ventral region of the tail-bud (right-arrowhead). (f) Transverse section of the head region in a stage 30 embryo indicated by the black bar in (b). Xprx-1 is expressed in the head mesenchyme (arrowheads).|
|Fig. 5. Expression of Xprx-1 in animal caps treated with peptide growth factors. Total RNA was isolated from animal caps treated with different concentrations of Activin A or bFGF for 6 h. RT-PCR and Southern blot analysis showed that Activin A dose dependently induced expression of Xprx-1 genes, but that bFGF did not. EF1-a served as an internal loading control. WE (whole embryo, stage 20) was a positive control, and RT- (reverse transcriptase not added to total WE RNA) was a negative control.|
|Fig. 6. Temporal expression of Xprx-1 in animal caps treated with Activin A. Animal caps were treated in Steinberg's solution with or without 100 ng/ml Activin A for 30 min. After treatment, the animal caps were maintained in normal Steinberg's solution for various periods (0-24 h). RT-PCR and Southern blot analysis showed induction of zygotic Xprx-1 expression after 6 h in Activin A-treated animal caps, but not in non-treated animal caps. EF1-a served as an internal loading control. WE (whole embryo, stage 20) is a positive control, and RT- (reverse transcriptase not added to total WE RNA) is a negative control.|
|prrx1 (paired related homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.|
|prrx1 (paired related homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior left, dorsal up.|